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CRYSTALLOGRAPHIC ANALYSIS OF THE SEC-DEPENDENT SECRETION CHAPERONE
CsaA
Yuliya Shapova B.Sc., Simon Fraser University, 2005
THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
In the Department of Molecular Biology and Biochemistry
O Yuliya Shapova 2007
SIMON FRASER UNIVERSITY
2007
All rights reserved. This work may not be reproduced in whole or in part, by photocopy
or other means, without permission of the author.
APPROVAL
Name:
Degree:
Title of Thesis:
Yuliya Shapova
Master of Science
Crystallographic Analysis of the Sec-dependent secretion chaperone CsaA.
Examining Committee:
Chair: Dr. David Vocadlo Assistant Professor, Department of Chemistry
Dr. Mark Paetzel Senior Supervisor Assistant Professor, Department of Molecular Biology and Biochemistry
Dr. Nancy Hawkins Supervisor Assistant Professor, Department of Molecular Biology and Biochemistry
Dr. Peter J. Unrau Supervisor Associate Professor, Department of Molecular Biology and Biochemistry
Dr. Jack Chen Internal Examiner Associate Professor, Department of Molecular Biology and Biochemistry
Date DefendedlApproved: u
SIMON FRASER ' UNIVERSITY~ brary
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Simon Fraser University Library Burnaby, BC, Canada
Revised: Spring 2007
ABSTRACT
The eubacterial protein CsaA has been proposed to act as a protein
secretion chaperone in the Sec-dependent translocation pathway. Two structures
of CsaA from Bacillus subtilis were solved by X-ray crystallography and refined to
1.9 and 2.0 A resolution. Structural analysis revealed two potential substrate
binding pockets on the surface of CsaA. These pockets display biochemical
properties consistent with the substrate binding preference of CsaA. A structure
of CsaA from Agrobacterium tumefaciens in complex with a phage display
derived peptide was solved to 1.65 A resolution. The peptide binds to the
substrate binding pocket on the surface of CsaA. Three residues of the bound
peptide form specific interactions with CsaA: glutamine at position (-6) and small
hydrophobic residues at positions (-5) and (-3). A conserved arginine residue in
the binding site of CsaA likely acts as a clamp that transiently interacts with and
stabilizes the peptides in the binding site.
Keywords: chaperone, X-ray crystallography, Secdependent protein secretion, peptide binding, protein structure
iii
First of all, I would like to thank my senior supervisor, Dr. Mark Paetzel, for
giving me a wonderful opportunity to learn about the exciting field of X-ray
crystallography and protein structure, and for his continuous support and
guidance along the way. I was greatly motivated by his interest and enthusiasm
for this project. I would also like to thank my supervisory committee members, Dr.
Nancy Hawkins and Dr. Peter Unrau, for their excellent advice and suggestions.
I would like to thank Dr. Anat Feldman for teaching me the techniques of
molecular biology and biochemistry, and Dr. David Oliver and Dr. Jaeyong Lee
for patiently answering my questions.
I would like to thank our lab manager, Deidre de Jong-Wong, for making
the Paetzel lab such an organized workplace, and for her support and
encouragement. Thank you also to all the past and present graduate students in
the Paetzel lab: Chuanyun Luo, Apollos Kim, Ivy Chung, Kelly Kim, Alison Li,
Charles Stevens, and Sung-Eun Nam, for creating such an enjoyable working
environment.
Finally, I would like to thank my husband, Oleg Titov, for always believing
in me, and for his unwavering support throughout my studies.
TABLE OF CONTENTS
Approval .............................................................................................................. ii ... Abstract .............................................................................................................. 111
Acknowledgements ........................................................................................... iv
Table of Contents ............................................................................................... v ... List o f Figures .................................................................................................. VIII
List of Tables ...................................................................................................... x
Glossary ............................................................................................................. xi
Chapter 1 . An Overview of the Molecular Chaperones from a ........................................................................................ Structural Perspective 1
Introduction ......................................................................................... 1 ..................................................................................... Trigger Factor 3
SurA and MPN555 ............................................................................. 6 ............................................................................... Hsp70 and Hsp40 7
Hsp9O ............................................................................................... 12 ............................................................................... Prefoldin (GimC) 14
Skp ................................................................................................... 17 LolA .................................................................................................. 18 PapD. FimC ...................................................................................... 20 Type Ill secretion chaperones .......................................................... 25 Signal Recognition Particle ............................................................... 28 SecB and CsaA ................................................................................ 33 TorD ................................................................................................. 36 GroEL and GroES ............................................................................ 38 Group II Chaperonins ....................................................................... 42 The ClplHspl00 family ..................................................................... 45 Conclusion ...................................................................................... 48
........... Chapter 2 . The Crystallographic Analysis of Bacillus subtilis CsaA 51 2.1. Introduction ....................................................................................... 51 2.2. Materials and Methods ..................................................................... 55
.......................................................................... 2.2.1. PCR and Cloning 55 ................................................... 2.2.2. Overexpression and Purification 58
2.2.3. Crystallization and Data Collection ............................................... 60 2.2.4. Structure Determination and Refinement ...................................... 61 2.2.5. Structural Analysis ........................................................................ 62
2.3. Results and Discussion .................................................................... 63
.......................................................................... 2.3.1. PCR and Cloning 63 ................................. 2.3.2. Overexpression and Purification of BsCsaA 66
2.3.3. Crystallization and Data Collection ............................................... 70 ...................................... 2.3.4. Structure Determination and Refinement 71
2.3.5. Sequence Alignment Analysis ....................................................... 74 ....................................................................... 2.3.6. Structural Overview 75
............................................................ 2.3.7. The Dimerization Interface 78 ............................................ 2.3.8. The Potential Substrate Binding Site 81
.............................. 2.3.9. The Electrostatics and Conservation Analysis 86 2.4. Conclusion ........................................................................................ 88
Chapter 3 . Cloning, Overexpression, Purification, Crystallization, and Refinement of the Crystal Structures of Agrobacterium tumefaciens CsaA .................................................................................................................. 89
3.1. Introduction ....................................................................................... 89 3.2. Materials and Methods ..................................................................... 91
.............................. 3.2.1. PCR and Cloning of AtCsaA and X15-AtCsaA 91 ....... 3.2.2. Overexpression and Purification of AtCsaA and XIS-AtCsaA 93
3.2.3. Crystallization and Data Collection of AtCsaA and X I 5- .......................................................................................... AtCsaA 94
3.2.4. Structure Determination and Refinement of AtCsaA and .................................................................................. X I 5-AtCsaA 95
........................................................................ 3.2.5. Structural Analysis 96 .................................................................... 3.3. Results and Discussion 97
......................................................... 3.3.1. PCR and Cloning of AtCsaA 97 .................................. 3.3.2. Overexpression and Purification of AtCsaA 99 ................................. 3.3.3. Crystallization of AtCsaA and X I 5-AtCsaA 101
3.3.4. Structure Determination and Refinement of AtCsaA and ................................................................................ X I 5-AtCsaA 103
3.3.5. An Overview of the AtCsaA structure and comparison to ....................................................................................... BsCsaA 105
......... 3.3.6. Interaction of X I 5-AtCsaA with the co-crystallized peptide 109 3.3.7. A comparison of the substrate binding pockets in the
structures of CsaA from A.tumefaciens, B.subtilis, and ............................................................................ T . fhermophilus 113
...................................................................................... 3.4. Conclusion 116
Appendix A . Cloning, Purification, and Crystallization of ....................................................................................... A . tumefaciens SecB 118 ..................................................................................... A.1. Introduction 118
................................................................... A.2. Materials and Methods 118 ........................................................................ A.2.1. PCR and Cloning 118
..................................... A.2.2. Protein Overexpression and Purification 119 ............................................. A.2.3. Crystallization and Data Collection 120
A.3. Results and Discussion ................................................................ 122 ........................................................................ A.3.1. PCR and Cloning 122
A.3.2. Overexpression and Purification of AtSecB protein .................... 124
A.3.3. Crystallization and Data Collection ............................................. 126
Appendix B ...................................................................................................... 128
Reference List ................................................................................................. 184
vii
LIST OF FIGURES
Figure 1 . 1 The structures of the Trigger Factor. SurA. and MPN555 ............... 5
Figure 1.2 The structures of Hsp70 and Hsp40 ......................................... 10
Figure 1.3 The structures of Hsp9O ................................................................ 13
Figure 1.4 The structures of prefoldin and Skp ......................................... 15
Figure 1.5 The structures of LolA and LolB .................................................... 20
Figure 1.6 The structures of the type I pili chaperone FimC and the P pili chaperone PapD ............................................................................ 23
Figure 1.7 The structures of the Type Ill secretion system chaperones in complex with their effector substrates .............................................. 26
Figure 1.8 The structures of the Signal Recognition Particle ......................... 30
Figure 1.9 The structures of SecB and CsaA ................................................. 35
Figure 1 . 10 The structure of TorD ................................................................. 37
Figure 1.1 1 The structures of GroES and GroEL .......................................... 40
Figure 1.12 The structure of the archaeal thermosome from Thermoplasma acidophilum ................................................................. 44
Figure 1 . 13 The structures of the Clp/Hsp100 family chaperones ClpA and ClpB .............................................................................................. 46
Figure 2.1 A schematic diagram of the Sec-dependent protein secretion in Gram-negative eubacteria ............................................................... 52
Figure 2.2 The optimization of the PCR amplification of B.subtilis csaA gene ..................................................................................................... 63
Figure 2.3 The results of cloning the PCR-amplified B.subtilis csaA gene into pCR2.1 -TOP0 vector ........................................................... 64
Figure 2.4 The results of subcloning of the csaA gene fragment into the expression vector pET28.a(+) ............................................................. 65
Figure 2.6 Purification of BsCsaA protein by nickel affinity chromatography ................................................................................... 67
Figure 2.7 Optimization of thrombin digest of BsCsaA ................................... 68
........... Figure 2.8 Purification of BsCsaA by size exclusion chromatography 69
viii
Figure 2.9 Crystals of B.subtilis CsaA ............................................................ 70
Figure 2.1 1 The structure of BsCsaA ............................................................ 76
Figure 2.12 Dimerization of BsCsaA via hydrogen bonding .......................................................................................... interactions 79
Figure 2.1 3 The potential substrate binding sites in BsCsaA ........................ 82
Figure 2.14 Docking of BsCsaA structure with a peptide in extended conformation ........................................................................................ 84
Figure 2.15 The conservation and surface electrostatic properties of BsCsaA ................................................................................................ 87
Figure 3.1 PCR amplification of A.tumefaciens CsaA gene ........................... 97
Figure 3.2 Cloning of A.tumefaciens CsaA .................................................... 98
.............. Figure 3.3 Purification of AtCsaA by nickel affinity chromatography 99
Figure 3.4 Optimization of the thrombin digest reaction of A.tumefaciens CsaA .......................................................................... 100
Figure 3.5 Initial crystals of AtCsaA ............................................................. 101
.............................................................. Figure 3.6 The structure of AtCsaA 107
Figure 3.7 The structure of AtCsaA in complex with a phage-display derived peptide (XI 5.AtCsaA) ........................................................... 110
Figure 3.8 The substrate binding pockets from the structures of AtCsaA. X I 5.AtCsaA. BsCsaA. and TtCsaA ..................................... 114
Figure A1 PCR and cloning of Ahmefaciens secB gene ............................ 122
Figure A2 Overexpression and purification of A.tumefaciens SecB ............ 124
Figure B1 A phylogenetic tree based on the sequences of 18 CsaA and 18 TRBP and MetRS (C-terminal part only) ....................................... 129
Figure B2 A sample diffraction pattern and a Ramachandran plot of the ............ crystallographic model of BsCsaA in the space group P3221 130
Figure 83 A sample diffraction pattern and a Ramachandran plot of the crystallographic model of BsCsaA in the space group P42,2 ............ 131
Figure 84 Ramachandran plots of the crystallographic models of AtCsaA ligand-free (A) and with the symmetry related in the putative binding site (B) .................................................................... 132
LIST OF TABLES
Table 2.1 The crystallographic data collection statistics for B.subtilis CsaA. . . . .. . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Table 2.2 A summary of refinement statistics for the models of B.subtilis CsaA structure. .............................. . . . . . . . . . . ........... ......................... 73
Table 2.3 The structural neighbors of B.subtilis CsaA .................................... 78
Table 2.4 The inter-chain hydrogen bonds between the two monomers of BsCsaA ...... . . .. . .. . . ... . .. . . .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Table 3.1 The data collection statistics for the structures of AtCsaA and X I 5-AtCsaA. .. .. .. . . . . ... . . .. . . .. . .. . . ... . .. .. . ... . . . . . .. . . . . . .. .. . . . . . . .. .. .. . . .. . . . . . .... . . . . . . . l o3
Table 3.2 The progress of refinement of AtCsaA and X I 5-AtCsaA structures ..... .. . . .. . . .. . .. . . .. . . .. . .. . ... . . .. ... . .. . .. . . .. . ... . . . . . . .. . .. . . .. .. .. . . . .. . .. . . .. . . .. . . lo4
Table 3.3 The refinement statistics for the structures of AtCsaA and X I 5-AtCsaA. .. . . .. ... . . .. . . .. . . . . . ... . .. .. .... . . .. . .. . . . . . .. .. . . . . . ... . . . . . . .. . . . . . ... . . . . . . . .. . . . lo5
Table 3.4 The interchain hydrogen bonds between the two monomers of AtCsaA. ...... . ... .. . . .. . . .. . . . . ... . . .. . ... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I08
Table 3.5 Direct hydrogen bonds between the peptide and X I 5-AtCsaA. .... 11 1
Table 61 Structures of the chaperone proteins listed in the Protein Data Bank. . . .. .. . . .. . .. . ... . .. . . .. . . .. . ... . .. .. ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
GLOSSARY
A Angstroms, a unit of measurement. 1A = lo-'' meters
ADP Adenosine diphosphate
Asymmetric unit The largest assembly of molecules that has no symmetry in itself, but can be superimposed on other identical elements in the unit cell by symmetry operations
ATP
B-factor
Adenosine triphosphate
A measurement of the displacement of an atom from its position due to thermal motion and conformational disorder. B-factor is elated to the displacement u by the equation:
= 8n2(u2\ High B-factors indicate a high degree of
disorder and a low degree of confidence about that particular part of the model.
Completeness The number of crystallographic reflections measured in a data set, expressed as a percentage of the total number of reflections present at the specified resolution.
Crystal An array of atoms, molecules, or ions, which are packed in a regularly ordered, repeating pattern extending in all three spatial dimensions.
Data
Dalton, a unit used to express molecular masses. 1 Da = 1 gram per mole
The positions and intensities of reflections from a single crystal in the diffraction pattern produced by X-ray crystallography
Electron Density An image of electron clouds surrounding the molecule Map
ER Endoplasmic reticulum
Hanging drop vapour diffusion
HEPES
l PTG
Macromolecular crowding
Mother liquor
Occupancy
PCR
PDB
PEG
Phage Display
A common method of protein crystallization, in which small volumes of precipitant and protein are mixed together and the drop is equilibrated against a larger reservoir of solution containing precipitant or another dehydrating agent. Drops are placed on a coverslip that seals the reservoir, such that they hang over the reservoir solution. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir until equilibration concentration is reached. This process may produce favourable conditions for crystallization.
4-(2-hydroxyethy1)-I-piperazineethanesulfonic acid
A significant volume of the cell cytosol is occupied by molecules (proteins, RNA, sugars and others). This crowding can drastically alter the kinetics or biophysical properties of molecules.
The part of a solution that is left over after the crystals are removed.
A measure of the fraction of molecules in the crystal in which a particular atom actually occupies the position specified in the model. If all molecules in the crystal are precisely identical, then occupancies for all atoms are 1.00.
Polymerase chain reaction
Protein Data Bank, http://www.rcsb.org
Polyethylene glycol
A technique used to select the peptide binding partners for the target protein. A library of variants of a peptide or protein is expressed on the outside of a phage virion. The selection process is carried out by incubating a library of phage-displayed peptides with a plate coated with the target, washing away the unbound phage, and eluting the specifically bound phage. The eluted phage is then amplified and taken through additional bindinglamplification cycles to enrich the pool in favour of binding sequences. After 3-4 rounds, individual clones are characterized by DNA sequencing and ELISA.
xii
Ramachandran A plot showing the main-chain conformational angles in a plot polypeptide. The conformational angles plotted are phi, the
torsional angle of the N-CA bond; and psi, the torsional angle of the CA-C bond. Due to steric repulsion, only certain conformational angles are allowed. The plot is used as a tool to assess the validity of the model.
Redundancy
Refinement
R factor
The data sets contain several independent measurements of each reflection due to symmetry in the crystal and overlap in measurements. Redundancy gives the average number of independent measurements of each reflection in a crystallographic data set and is calculated as: (number of measured reflections) 1 (number of unique reflections).
The process of improving the agreement between the molecular model and the crystallographic data by adjustment of positions, occupancies, and B-factors of atoms in the model. Progress in refinement is signified by decreasing R values, disappearance of residues from the unfavourable region of the Ramachandran plot, and improving chemical plausibility of the structure (e.g. bond lengths and angles).
A measure of agreement between the crystallographic model and the original X-ray diffraction data, calculated as
R = C I l ~ ~ ~ ~ 1 - IFc&II/CIFobsI where ~~b~ and F ~ ~ I ~ are intensities observed from the measured data or calculated from the model, respectively.
Calculated using the same equation as the R-factor, except only for a small subset (5-1 0%) of randomly chosen intensities, which are set aside from the beginning and not used in refinement. Rkee measures how well the current model predicts a subset of the measured reflection intensities that were not included in the refinement. Rfree is higher than the R factor at the beginning of refinement, but in the final stages, the two values should become more similar.
A measure of agreement among multiple measurements of the same reflections, with the different measurements being in different frames of data or different data sets. R,,,,, is calculated as follows: Rm,, = C 11 - (I)I/Z ( I ) (I is the
individual measurement of each reflection, and <I> is the
xiii
average intensity from multiple observations):
r.m.s.d. Root mean square deviation
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Sitting drop Same principles as described for hanging drop vapour vapour diffusion diffusion, except drops are placed on a pedestal above the
reservoir solution
Space group Designation of the symmetry of the unit cell of a crystal.
TRlS 2-amino-2-hydroxymethyl-1,3-propanediol
Unit cell The simplest repeating unit in the crystal that is representative of the entire crystal
xiv
CHAPTER 1. AN OVERVIEW OF THE MOLECULAR CHAPERONES FROM A STRUCTURAL PERSPECTIVE
1 .I. Introduction
Originally, the term "molecular chaperone" was used to describe a class of
proteins that assist the correct folding and assembly of other polypeptides but are
not part of the final structure (Ellis, 1987). Even though is has been demonstrated
that most proteins can assume their correct fold in vifro without the assistance of
any other factors (Anfinsen, 1973), the situation in the cell is quite different
because of the presence of numerous other proteins that produce the effect of
macromolecular crowding (Ellis & Minton, 2006). In these conditions, it may be
problematic for proteins to reach their correct fold. Therefore, many proteins rely
on molecular chaperones to create a proper microenvironment that produces
favourable conditions for their folding and prevents improper interactions
(Buchner & Walter, 2005, Ellis & Minton, 2006, Hartl, 1996).
The heat shock proteins, or hsps, are a large and diverse category of
molecular chaperones whose expression is enhanced during the conditions of
cellular stress. These proteins function to prevent protein aggregation during
stress conditions and increase the cell survival. However, it was subsequently
discovered that these proteins also have housekeeping functions, such as de
novo protein folding, resolubilization of protein aggregates, or protein degradation
((Buchner & Walter, 2005, Hartl, 1996, Hartl & Hayer-Hartl, 2002, Young et al,
2004). Besides hsps, numerous other chaperones had been discovered that
function in specific pathways and participate in a diverse range of activities, such
as assembly of oligomeric proteins or transport (Leroux, 2001). The general
function of these molecular chaperones is to bind to and stabilize unstable
conformers of their substrates in order to ensure their correct cellular fate (Hartl,
1996). Since chaperones are found in all three kingdoms of life and often are
essential for the cell survival, they are an important part of the proper functioning
of the cell (Leroux, 2001).
The three-dimensional structures of molecular chaperones provide
invaluable information on how these proteins are able to carry out their functions.
The structural basis of the chaperone-substrate interaction is especially
important, as it may reveal common strategies in the mechanism of the
chaperone action. This chapter discusses the structures of several molecular
chaperones with a special emphasis placed on the structural features that allow
them to interact with their substrates. The goal of this chapter is to identify
unifying schemes in the structures of molecular chaperones and the chaperone-
substrate interactions.
1.2. Trigger Factor
Trigger factor (TF) is the first chaperone to associate with the nascent
polypeptide just as it comes out of the ribosomal peptide exit tunnel (Hesterkamp
et al, 1996) and is found exclusively in eubacteria (Leroux, 2001). Trigger factor
associates with the large subunit of the bacterial ribosome in a 1:l ratio; the
association occurs through the ribosomal L23 protein located near the peptide
exit tunnel (Ferbitz et al, 2004, Kramer et al, 2002). TF works in cooperation with
other chaperones that associate with the newly synthesized polypeptide
downstream of TF, such as DnaK, GroEL, SecB, and the signal recognition
particle (SRP) (Deuerling et al, 1999, Ha et al, 2004, Kandror et al, 1995, Ullers
et al, 2006). TF and DnaK have overlapping substrate specificities (Deuerling et
al, 2003), and disruption of both DnaK and trigger factor genes causes massive
protein aggregation and is lethal to the cell (Deuerling et al, 1999). Unlike DnaK,
TF is ATP-independent and does not appear to be able to prevent the
aggregation of thermally denatured proteins in vitro (Schaffitzel et al, 2001). The
ribosome-bound TF prevents the proteinase K - induced degradation of large (up
to 41 kDa) stretches of unfolded polypeptides emerging from the ribosome
(Hoffmann et all 2006). The substrate binding specificity of TF was analyzed by
scanning cellulose-bound peptide libraries representing overlapping 13-mer
sequences of five Exoli proteins. TF prefers to bind eight residue stretches of
aromatic and basic amino acids with a positive net charge without positional
preference (Patzelt et al, 2001).
Several structures of TF are available, including a full-length structure
from Escherichia coli in complex with the ribosome (Ferbitz et all 2004), as well
as several other structures of the TF fragments alone or in complex with the
bacterial ribosomal proteins. The structure of TF is composed of three
functionally distinct domains: the N-terminal domain is responsible for the
interactions with the ribosome (Hesterkamp et al, 1996), and the middle domain
contains peptidyl-prolyl cisltrans isomerase (PPlase) activity (Maier et al, 2005).
The C-terminal domain has recently been demonstrated as the central module of
the chaperone activity (Merz et all 2006). Surprisingly, the three-dimensional
organization of these domains does not resemble the linear sequence, since the
C-terminal domain is located in the middle of the structure, between the N-
terminal and PPlase domains, which are located at the opposite ends of the
structure and do not interact with each other (Figure 1.1) (Ferbitz et all 2004,
Ludlam et all 2004). All interactions with the ribosome are accomplished by the
N-terminal domain through the conserved GFRxGxxP motif (Kramer et al, 2002)
located in the loop between the two a-helices (Ludlam et al, 2004).
The PPlase domain, which has been demonstrated as dispensable for the
chaperone activity (Kramer et al, 2004), resembles a well-described FKBP fold
found in several other PPlases (Ludlam et all 2004). The C-terminal domain is
composed of several a-helices that form two extended "arms" and is structurally
similar to the periplasmic chaperone SurA (Ferbitz et all 2004). TF is tethered at
the ribosome via its N-terminal domain, and the C-terminal domain is positioned
such that its "arms" hover over the ribosomal peptide exit side. The complete
Figure 1.1 The structures of the Trigger Factor, SurA, and MPN555.
A) A cartoon diagram of the Trigger Factor from Escherichia coli, PDB ID 1W26 (Ferbitz et al, 2004). The N-terminal domain is in green, the PPlase domain is in red, and the C-terminal domain is in blue. B) The protein surface electrostatics of Trigger Factor, PDB ID 1W26. Areas coloured white, red, and blue, correspond to neutral, negative, and positive surface electrostatics potential, respectively. Arrows indicate the location of the "arms" in the C-terminal substrate binding site C) Cartoon diagram of MPN555 from Mycoplasma pneumoniae (IZXJ, (Schulze-Gahmen et al, 2005)). D) Cartoon diagram of SurA from E.coli (lM5Y. (Bitto & McKay, 2002)). The PPlase domains are in red, and the substrate binding domain is in blue. E) The protein surface electrostatics of SurA. The peptide from a symmetry related molecule is shown as green cartoon. Arrows indicate the "arms" of the substrate binding domain. F) A side view of the substrate binding domain in (E), showing the peptide binding into the groove between the two "arms". The figures were made using PyMol (DeLano, 2002).
structure adopts a "crouching dragon" conformation, with a large "cradle"
between the N-terminal domain and the C-terminal "arms", which can
accommodate folded protein domains as large as 14 kDa (Ferbitz et all 2004).
The interior of the "cradle" is very hydrophobic. It has been suggested that this
"cradle" shields the newly synthesized polypeptides from the environment and
delays folding until a sufficient stretch of protein sequence is synthesized for a
protein domain to fold correctly (Ferbitz et al, 2004).
1.3. SurA and MPN555
SurA is a periplasmic, ATP-independent molecular chaperone that
promotes the folding and maturation of outer membrane porins, such as LamB,
OmpF, and OmpA (Behrens et all 2001, Hennecke et all 2005). SurA is selective
for its substrates, and prefers to bind to sequences enriched in aromatic residues
(Hennecke et al, 2005). Like trigger factor, SurA exhibits modular architecture.
SurA contains two FKBP-like PPlase domains, both of which are dispensable for
its chaperone activity, and a bipartite core domain composed of N- and C-
terminal sequences, which is responsible for the chaperone activity (Behrens et
al, 2001, Bitto & McKay, 2002). The catalytic domain P2 is located away from the
rest of the structure and is tethered to the rest of the protein via linkers, whereas
the second PPlase domain, PI , which is not catalytically active on its own, is
located close to the chaperone core domain (Figure 1.1) (Bitto & McKay, 2002,
Bitto & McKay, 2002). The substrate-binding domain is similar in fold to the C-
terminal domain of the trigger factor and to MPN555, a protein of unknown
function from Mycoplasma pneumoniae (Figure 1.1) (Schulze-Gahmen et al,
2005). It is composed of a helices which form two arm-like projections with a 50
14 hydrophobic crevice in between (Bitto & McKay, 2002). In the crystal structure
of SurA published by Bitto et a/, this hydrophobic crevice contains a peptide from
a neighbor molecule, which identifies it as a site of substrate binding (Figure 1 .I).
The peptide binds in an a-helical conformation between the arms of the core
domain. The inner surfaces of these arms contain hydrophobic pockets, which
interact with Leu153 and Val157 residues on the peptide. There are patches of
negative charge in the bottom of the crevice and on the inner surface of the
arms, which may provide potential for selecting specific substrates. The bound
peptide is only 15 14 long, but the crevice can potentially accommodate longer
peptides (Bitto & McKay, 2002).
1.4. Hsp7O and Hsp40
Hsp70 is the central component of the cellular system of molecular
chaperones; its homologues are found in almost all organisms, including
eubacteria, most archaea, and the cytosol and organelles of eukaryotes. The
general functions of this ATP-dependent chaperone include de novo folding of
polypeptides, prevention of protein aggregation, and refolding of aggregated
proteins (Mayer & Bukau, 2005a). In addition to playing a key role in the heat
shock response, hsp70 acts as a housekeeping protein during non-stress
conditions and participates in a wide variety of cellular processes, such as signal
transduction, protein translocation across membranes, protein quality control,
and interaction with regulatory proteins (Mayer & Bukau, 1998). The bacterial
hsp70 homolog DnaK has been estimated to assist the de novo folding of 10-
20% of newly synthesized polypeptides (Hartl & Hayer-Hartl, 2002). Most cells
encode multiple homologues of Hsp70 that are conserved in sequence yet carry
out diverse cellular roles. Almost all Hsp70 proteins interact with a J-domain co-
chaperone, such as hsp40 in the eukaryotic cytosol or DnaJ in E.coli, to stimulate
their intrinsically low ATPase activity and regulate interactions with substrates
(Hennessy et al, 2005). The J-domain, a highly conserved structure found at the
N-terminus of hsp40, is required for interaction with hsp70, however, many hsp40
proteins contain additional domains that allow them to carry out other specific
tasks (Qiu et all 2006). For example, some hsp40 proteins have their own
chaperone activity and form transient, ATP-independent associations with the
client proteins (Qiu et all 2006). In E.coli, which contains three hsp70 homologs
and six hsp40 homologs, there is a specific pattern of hsp701hsp40 interactions
whereby a particular hsp70 protein may have one or multiple hsp40 partners (Qiu
et all 2006).
Hsp70*ATP has low affinity and fast exchange rates for substrates,
whereas Hsp70*ADP has high affinity and slow exchange rates (Laufen et al,
1999). The following reaction cycle has been suggested for the E.coli hsp70
(DnaK). The substrate bound to DnaJ (an hsp40 homolog) is transferred to
DnaK*ATP. DnaJ also modulates the communication between the ATPase and
the substrate-binding domains of DnaK. Both DnaJ and substrate binding
stimulate the ATP hydrolysis by hsp70, which locks the substrate into the binding
cavity of hsp70. The substrate dissociates from hsp70 upon exchange of ADP for
ATP (Laufen et al, 1999, Young et all 2004).
In addition to hsp40, hsp70 recruits other proteins, such as nucleotide
exchange factors GrpE (in E.coli) and BAG-1 (in eukaryotes), as well as Hip,
which is thought to stabilize the ADP-bound state of hsp70; Hop, an hsp70-hsp90
organizing protein; and CHIP, which may connect hsp70 to the protein
degradation pathway (Mayer & Bukau, 2005b).
Hsp70 consists of a 45 kDa N-terminal ATPase domain (NBD) and a 25
kDa substrate binding domain (SBD) (Figure 1.2) (Mayer & Bukau, 2005a). The
ATPase and the substrate binding domains are connected by a linker of -10
residues long that may be responsible for the interdomain communication, which
couples substrate binding to ATP hydrolysis (Vogel et al, 2006). Numerous
structures of the separate ATPase and substrate binding domains of hsp70 are
available, as well as the structure of a functionally intact, nearly full-length bovine
hsc70 (a constitutively induced hsp70 homolog) that further highlights the role of
the interdomain linker in the communication between NBD and SBD (Jiang et all
2005). The N-terminal ATPase domain consists of two subdomains which form
two lobes with a deep cleft between them wherein the nucleotide binds (Flaherty
et al, 1990, O'Brien et all 1996). The substrate binding domain can be further
subdivided into a P-sandwich subdomain and a C-terminal a-helical subdomain
(Zhu et al, 1996) The structure of the SBD from E.coli DnaK in complex with a
synthetic peptide NRLLLTG reveals that the peptide binds only to the P-sandwich
subdomain, whereas the a-helical subdomain serves as a lid that locks the
substrate into place (Figure 1.2) (Zhu et al, 1996). The peptide binds in an
extended conformation and contacts DnaK through side-chain mediated van der
biz-
&-*- I-'
Figure 1.2 The structures of Hsp70 and Hsp40.
A) A cartoon diagram of bovine hsc70, an hsp70 hornologue. The nucleotide binding domains of hsc70 from PDB entries IYUW (Jiang et al, 2005) (in olive) and IKAX (O'Brien et al, 1996) (in purple) were superimposed with the r.m.s.d. of 1.38 A over 378 C, atoms. The program Superpose (Maiti et al, 2004) was used for superposition. ATP from entry IKAX is shown as orange spheres. The SBD from PDB entry 1YUW is in blue and lacks the 100 C-terminal residues. B) The substrate binding domain (blue, cartoon)of E.coli DnaK in complex with a peptide (green, spheres); PDB ID IDKX (Zhu et al, 1996). C) Left: as in B), except the SBD is coloured according protein surface electrostatics, and the peptide is shown as sticks. Right: a close-up view of the area bounded by a box in the left pane. The leucine binding pocket is indicated by an arrow. D) Left: A cartoon diagram of the C- terminal fragment of Ydjl, a Saccharomices cerevisae hsp40 homologue (green) in complex with a peptide (magenta, sticks). PDB ID INLT (Li et al, 2003). Right: a close-up view of the area bounded by a box in the left pane. The leucine binding pocket is indicated by an arrow. The surface of Ydj is coloured according to protein surface electrostatics, and the peptide is shown as sticks.
Waals interactions and main-chain hydrogen bonds. (Figure 1.2BC). A deep
hydrophobic pocket in the DnaK substrate binding site buries a leucine side chain
from the peptide and is important for substrate specificity. The dimensions and
biochemical characteristics of this pocket are ideal to bind leucine, but it may also
accommodate methionine, isoleucine, or smaller side chains. Bulky hydrophobic,
charged, or polar residues would be disfavoured at this position (Zhu et al, 1996).
The substrate binding site on DnaK is generally hydrophobic in nature, and has
an area of a slightly negative electrostatic potential adjacent to it (Zhu et al,
1996). This is consistent with the fact that DnaK prefers to bind stretches of four
to five hydrophobic residues flanked by basic residues, whereas acidic residues
are disfavoured (Rudiger et al, 1997).
The structure of the substrate binding domain of yeast hsp40 in complex
with a peptide substrate has been reported by Li et a1 (Figure 1.3) (Li et al, 2003).
The domain responsible for peptide binding consists of two P-sheets connected
by a short helix. The peptide GWYLEIS binds into an open hydrophobic groove
on the hsp40 surface by complementing one of the P-sheets as an antiparallel P-
strand. The chaperone-substrate interactions are similar to that seen in DnaK,
since they involve the peptide binding in an extended conformation via main-
chain hydrogen bonds and van der Waals contacts (Figure 1.2D). Importantly, a
central leucine residue on the peptide is buried in a hydrophobic pocket of hsp40,
analogous to that seen in the structure of hsp70 in complex with peptide (Li et al,
2003). This correlates well with the fact that hsp40 transfers the bound
substrates to hsp70, which would require the ability to bind to the same
substrates.
1.5. Hsp9O
The 90 kDa heat shock protein (hsp90) is a highly conserved and
abundant protein found in the cytosol of eubacteria and in the cytosol,
mitochondria, ER, and chloroplasts of eukaryotic cells (Picard, 2002). It is
essential in eukaryotes, and participates in folding and stabilization of specific
client proteins involved in a multitude of cellular processes, such as signal
transduction, transport, transcription, cell cycle regulation, and protein quality
control (Zhao & Houry, 2007). In cooperation with hsp70140, hsp90 may act in
refolding of misfolded proteins during stress conditions, however, it has not been
implicated in de novo protein folding (Mayer & Bukau, 1999). Hsp9O is ATP-
dependent and relies on various co-chaperones to regulate its ATPase cycle and
interactions with substrates (Pearl & Prodromou, 2006). For example, hsp90
cooperates with hsp70140 through an adaptor protein Hop to assist in folding of
steroid hormone receptors (Hernandez et all 2002), whereas the co-chaperone
cdc37 (p50) is recruited for interactions with the protein kinase clients (Roe et all
2004).
There are numerous structures of hsp90 available in the Protein Data
Bank (Berman et all 2000), including complexes with inhibitors and co-
chaperones. The overall structure of Hsp9O is modular and consists of three
domains arranged in a linear fashion: the N-terminal domain is responsible for
ATP and drug binding, the middle domain has been implicated in substrate
12
Figure 1.3 The structures of Hsp9O.
A) The closed conformation of Hsp9O. A cartoon diagram of Saccharomices cerevisae Hsp82, an Hsp9O homologue, in complex with ATP (2CG9, (Ali et al, 2006)). B) The open conformation of Hsp9O. A cartoon diagram of the nucleotide- free E.coli HtpG, an Hsp9O homologue (210Q, (Shiau et al, 2006)). In both A) and B), the N-terminal nucleotide binding domains are in green, the middle domains are in blue, and the C-terminal dirnerization domains are in red. ATP bound to the N- terminal domain in A) is in purple.
binding as well as contributing to the ATPase activity through a key catalytic
arginine residue, and the C-terminal domain is essential for Hsp9O dimerization
(Figure 1.3) (Pearl & Prodromou, 2006).
Two full-length hsp90 structures have been published recently: the yeast
hsp90 in complex with an ATP analogue and a co-chaperone p23lSbal (Ali et al,
2006), as well as an E.coli hsp90 homolog, HtpG, alone or in complex with ADP
(Shiau et al, 2006). These structures represent the three different nucleotide-
induced conformations of hsp90. In the nucleotide-free state, the dimeric hsp90
adopts an open, highly flexible V-shaped conformation. The two monomers
dimerize at the C-terminal domains via a four-helix bundle; a large hydrophobic
cleft between the two monomers is proposed to be the site of the substrate
binding (Figure 1.3B) (Shiau et al, 2006). Upon nucleotide binding, hsp90
undergoes significant conformational rearrangements and assumes a "closed"
state via the dimerization of the N-terminal domains (Figure 1.2A) (Ali et al,
2006). In the hsp90 structure in complex with a non-hydrolysable ATP analogue,
the two N-terminal domains form a stable dimer via the exchange of N-terminal
P-strands and hydrophobic interactions, and the middle domains are brought
close together (Ali et al, 2006). Although it has been proposed earlier that hsp90
encloses the client proteins in its central cleft acting as a "molecular clamp"
(Meyer et al, 2003b) , Ali et a1 demonstrated that in the ATP-bound "closed"
state, the dimeric cleft becomes too narrow to accommodate a folded substrate
(Ali et al, 2006). At present, it is not clear exactly how hsp90 binds its substrates,
necessitating further studies and perhaps determining the structure of hsp90 in
complex with a client protein to address this question.
1.6. Prefoldin (GimC)
Prefoldin is a heterohexameric chaperone found in archaea and in
eukaryotic cytosol, but not in eubacteria. Prefoldin binds to non-native proteins in
an ATP-independent manner and prevents them from aggregation. Prefoldin then
transfers its substrate to the class II chaperonin (eukaryotic CCT or archaeal
thermosome), thus acting as a co-chaperonin (Okochi et al, 2004, Vainberg et al,
1998). In eukaryotes, prefoldin seems to be specialized for binding cytoskeletal
proteins actin and tubulin, whereas in archaea, which lacks actin and tubulin, it
may play a more general role in protein folding similar to that of hsp7O (Leroux,
Figure 1.4 The structures of prefoldin and Skp.
A), B), C), and F) are based on the structure of archaeal prefoldin from Methanobacterium thermoautotrophicum. PDB ID 1FXK (Siegert et al, 2000). A) A cartoon diagram of the a-class prefoldin subunit. B) P-class prefoldin subunit. C) The prefoldin hexamer, side view. a-subunits (orange) are located in the middle of the structure and serve as the central points for oligomerization of the P-subunits (green). D) and E) are based on the structure of E.coli Skp, PDB ID 1SG2 (Korndorfer et al, 2004). D) A monomer of Skp. E) Skp trimer, side view. F) Surface representation of prefoldin, view from the bottom, coloured according to the surface vacuum electrostatics.
2001). The prefoldin binding site in actin and tubulin is signified by the motif
EHGl preceded by several hydrophobic residues (Rommelaere et all 2001).
The prefoldin hexamer is composed of two types of prefoldin subunits:
class a and class p. Archaea possess only one homolog of each class, whereas
eukaryotes have two class a homologs and four class P homologs (Siegert et al,
2000). The crystal structure of the archaeal prefoldin was solved by Siegert et a1
in 2000. The individual structures of the a- and P-class subunits are similar in that
they have two long a-helices at the N-terminus and C-terminus, which fold over
to make a coiled coil. The central part of each subunit contains one (in p-class) or
two (in a-class) P-hairpin structures, which are responsible for oligomerization by
forming a central platform of two P-barrels (Figure 1.4). The overall structure of
the assembled prefoldin hexamer resembles a jellyfish with the central P-barrels
serving as the "body" from which six flexible coiled coil "tentacles" extend (Figure
1.4) (Siegert et al, 2000). The coiled coils are partially untwisted at their tips and
expose hydrophobic patches that are crucial for interaction with the substrates
(Okochi et all 2004, Siegert et al, 2000). An additional region of hydrophobicity is
located at the bottom of the cavity formed by the six prefoldin coiled coil
"tentacles" and has been proposed to protect the unfolded substrates from the
cytosolic environment (Siegert et al, 2000). The complex between eukaryotic
prefoldin and actin as revealed by electron microscopy shows an unfolded actin
binding into the central cavity of prefoldin and interacting with the tentacle tips
(Martin-Benito et all 2002). In addition, prefoldin was shown to bind to the apical
domains of one or both CCT rings via the tips of its tentacles (Martin-Benito et al,
2002). In prefoldin, the binding sites for the chaperonin appear to be adjacent to
the peptide-binding sites, which may be important for efficient substrate transfer
(Okochi et all 2004).
1.7. Skp
Skp is a periplasmic molecular chaperone of Gram-negative bacteria that
is involved in biogenesis of outer membrane porins, such as OmpA, OmpF,
OmpC, and LamB (Chen & Henning, 1996). The proteins that are being
translocated across the inner membrane in an unfolded state are prone to
aggregation immediately upon emergence in the periplasm. Skp binds to its
target proteins at the inner membrane as they emerge from the Sec translocon,
thereby protecting them from misfolding and aggregation in the periplasm (Harms
et all 2001). In addition, Skp facilitates the insertion of its substrates into the
outer membrane, a function that requires Skp interaction with a
lipopolysaccharide (Bulieris et all 2003). Two other periplasmic chaperones,
SurA and DegP, have been implicated in interactions with outer membrane
proteins. On the basis of the analysis of null mutations of these three
chaperones, it has been suggested the periplasm of E.coli contains two parallel
pathways for folding and insertion of outer membrane proteins, where Skp and
DegP are part of one pathway, and SurA is part of a separate pathway (Rizzitello
et all 2001).
Two crystal structures of E.coli Skp are currently available in the PDB
database. The structure of the monomeric Skp can be divided into two
subdomains: the core subdomain and the a-helical extensions from the core. The
17
core subdomain is composed of sequences located at the N- and C-termini and
contains two short a-helices and four @-strands (Walton & Sousa, 2004). This
core subdomain is responsible for the formation of the functional Skp
homotrimer. The middle part of each monomer contains two long a-helices joined
by a loop, which run along one another in an antiparallel fashion (Figure 1.4)
(Walton & Sousa, 2004). The core subdomains from each monomer associate
via their @-strands to form three inter-subunit @-sheets around the central 3-fold
axis. The a-helical subdomains are flexible and project away from the trimeric
core (Korndorfer et al, 2004). The inside surfaces of the a-helical "tentacles" are
hydrophobic in character and are arranged around the central cavity, which is a
plausible site for substrate binding (Korndorfer et al, 2004, Walton & Sousa,
2004). The tip of each "tentacle" contains a region of positive charge, which is
different from prefoldin, where the tentacle tips are hydrophobic and are involved
in substrate binding (Korndorfer et al, 2004).
Strikingly, the assembled structure of Skp resembles the jellyfish shape
with a-helical "tentacles", which is also seen in prefoldin, in spite of the different
topology of the secondary structural elements and no sequence similarity
between the two proteins. (Figure 1.4) (Korndorfer et all 2004). It is therefore
possible that the structural and functional similarities between prefoldin and Skp
arose via convergent evolution (Korndorfer et all 2004).
1.8. LolA
The periplasmic chaperone LolA is part of the E.coli Lol system
responsible for the sorting and localization of lipoproteins to the outer membrane.
18
Mature lipoproteins in the periplasm contain an N-terminal cysteine residue
modified with a lipid moiety (Tokuda & Matsuyama, 2004). Lipoproteins that are
destined to remain in the inner membrane contain an aspartic acid residue at
position 2, which serves as Lol avoidance signal (Tokuda & Matsuyama, 2004).
Other components of the Lol system include LolCDE, an ATP-binding cassette
(ABC) transporter anchored in the inner membrane, and LolB, a lipoprotein
receptor in the outer membrane. LolCDE releases the lipoproteins from the inner
membrane, transferring them to the chaperone LolA, which carries the
lipoproteins across the periplasmic space and transfers them to LolB in the outer
membrane. The lipoprotein transfer from LolA to LolB is ATP-independent and
occurs because LolB has higher affinity to lipoproteins than that of LolA (Tokuda
& Matsuyama, 2004).
The crystal structure of LolA from E.coli is available in the Protein Data
Bank (Takeda et al, 2003). LolA consists of an I I-stranded antiparallel P-sheet
forming un unclosed P-barrel, and 3 a-helices located on one face of the sheet
(Figure 1.5A). The inner surface of the P-sheet contains a hydrophobic cavity that
may act as a binding site for the lipid moieties on lipoproteins. The three a-
helices are also hydrophobic in character and act as a lid to the putative binding
site (Takeda et al, 2003). The lid is "locked" in place via interactions between
arginine 43 of the P-sheet and the residues in the a-helices, but is expected to
open and close upon binding and release of lipoproteins (Takeda et al, 2003). A
recent study which involved mutating the residues forming the hydrophobic cavity
and the a-helical lid demonstrated that both these regions are crucial for binding
Figure 1.5 The structures of LolA and LolB.
A) The structure of LolA chaperone (liwl, (Takeda et al, 2003)) in cartoon representation. B) The structure of LolB receptor in complex with PEG 2000 MME (IIWN, (Takeda et al, 2003)). LolB is shown as pink ribbon, and PEG2000 MME as green sticks.
lipoproteins and their transfer to LolB (Watanabe et al, 2006).
Interestingly, the structure of LolB receptor is strikingly similar to that of
LolA chaperone, despite the low sequence identity of 8%. In contrast to LolA,
however, the a-helical lid of LolB is in an open conformation, and the putative
binding site accommodates a molecule of polyethylene glycol 2000 monomethyl
ether (PEG 2000 MME), a compound used to crystallize the protein (Figure 1.5B)
(Takeda et all 2003)
1.9. PapD, FimC
Gram-negative pathogenic bacteria utilize thread-like extracellular
adhesive projections termed pili in order to recognize and interact with their
specific receptors on the surface of the host cells. Pili are important virulence
factors and are responsible for a wide variety of infectious diseases (Piatek et al,
2005). The E.coli Type 1 and P pili, as well as over 30 other pili and non-pili
structures from various Gram-negative pathogens, are assembled in the
periplasm via the chaperone-usher pathway (Sauer et all 2000). Both type 1 and
P pili have similar heteropolymeric structure, and are encoded by the E.coli fim
and pap gene clusters, respectively. They are rigid helical rods assembled from
identical pilin subunits, FimA in type 1 and PapA in P pili. Each rod is joined to a
thinner tip fibrillum, made up of other types of fim or pap subunits, followed by an
adhesin subunit at the distal end. Two proteins are responsible for the pili
assembly: a soluble periplasmic chaperone (FimC in type 1 and PapD in P pili)
and an usher (FimD in type 1 and PapC in P pili), integrated into the outer
membrane (Capitani et all 2006, Sauer et all 2000).
FimC and PapD chaperones are responsible for binding the fim or pap
type pilin subunits, respectively, as they emerge from the Sec translocon, and
targeting them to their respective ushers, which add the subunits to the growing
pilus fiber on the outer cell surface. The subunit structure consists of an
incomplete immunoglobulin-like (lg) fold, missing the seventh, C-terminal strand.
In the assembled pilus structure, this seventh strand is supplied by a highly
conserved N-terminal extension upstream of the lg fold in an adjacent subunit
(Sauer et al, 2002). The interactions of the pili subunits with the chaperone and
the usher proceed in an ATP-independent manner.
In the absence of the chaperone, the pilus subunits do no fold properly
and aggregate (Barnhart et all 2000). The chaperone binding stabilizes the
subunit and prevents it from aggregation or premature interactions with other pili
subunits (Kuehn et al, 1991). The structures of FimC and PapD chaperones are
very similar (Piatek et all 2005). The chaperones consist of two 19-like domains,
with each domain containing seven antiparallel P-strands divided into two P-
sheets that pack against one another. The two domains are arranged at an angle
to one another, such that the complete structure resembles a boomerang. The
two domains are joined by a -30 residue long linker, enriched in hydrophobic
amino acids (Holmgren & Branden, 1989).
Several structures of FimC and PapD chaperones in complex with their
respective pilus subunits have been obtained. These structures reveal the basis
of the chaperone-subunit interaction. Unlike many cytoplasmic chaperones, FimC
and PapD bind their substrates in a native-like state (Figure 1.6) (Kuehn et al,
1991). The chaperone stabilizes the subunit via the donor-strand
complementation mechanism, which involves donating one of its own strands to
complete the lg fold of the pilus subunit (Choudhury et all 1999, Sauer et al,
1999). In the PapD-PapK structure, the chaperone PapD inserts its strand G I
into a hydrophobic groove between the strands A and F of PapK (Figure 1.5AB).
The inserted strand makes parallel P-sheet interactions with the subunit strand F,
thus creating a non-canonical lg fold (Sauer et all 1999). This is in contrast to the
pili subunit-subunit complexes, where the N-terminal extension from one subunit
complements the F strand of another subunit in an antiparallel fashion, making a
more stable, canonical lg fold (Sauer et al, 2002). Since the pili subunit-subunit
interactions are more stable than that of chaperone-subunit, the displacement of
the chaperone G I strand in the subunit groove by the N-terminal extension of
Figure 1.6 The structures of the type 1 pili chaperone FimC and the P pili chaperone PapD.
A) and 6) are based on the structure of PapD in complex with a pilin subunit PapK (PDB ID IPDK (Sauer et al, 1999)). A) A cartoon diagram of PapD (green) and PapK (cyan). The PapD strand G I forms a parallel P-sheet with the PapK strand F. B) PapK is shown as surface coloured according to the electrostatic potential. The G I strand of PapD is shown as sticks. C) and D) are based on the structure of FimC in complex with the adhesin subunit FimH (IQUN (Choudhury et al, 1999)). The N- terminal lectin domain of FimH is not shown for clarity. C) A cartoon diagram of FimC (orange) and FimH (purple). D) FimH is shown as surface coloured according to the electrostatic potential. The GI strand of FimC is shown as sticks.
another subunit may be energetically favourable and may drive the fiber
formation (Sauer et all 2002).
The chaperone G1 strand has a pattern of alternating hydrophobic
residues, which is also found in the N-terminal extensions of the pilus subunits
(Sauer et all 1999). The G1 strand interacts with the residues of the groove via
main-chain hydrogen bonds to strands A and F, as well as hydrophobic
interactions with the base of the groove. In addition, there are several side-chain
mediated hydrogen bonds between the chaperone and the C-terminus of the
subunit that serve to anchor the subunit into the cleft between the two domains of
the chaperone (Sauer et all 1999). A very similar structure and the mechanism of
donor strand complementation were reported for the FimC-FimH complex (Figure
1.6CD) (Choudhury et all 1999).
Several roles were suggested for the chaperone in the pili biogenesis
pathway. First, the chaperone stabilizes the pili subunit and prevents it from
unproductive aggregations by capping the hydrophobic groove in the subunit
(Choudhury et all 1999, Sauer et all 1999). Second, it keeps the subunit primed
for displacement of the chaperone strand G1 by the N-terminal extension of
another subunit, necessary for assembly into the pilus fiber (Sauer et all 2002).
Third, it may facilitate the folding of the subunits in the periplasm, possibly by
providing the missing steric information necessary for correct folding of the
subunit (Barnhart et al, 2000, Vetsch et all 2004).
I . lo. Type Ill secretion chaperones
Many Gram-negative pathogenic bacteria interact with the host cells by
directly injecting their virulence factors into the host cytosol via the type Ill
secretion machine, a needle-like structure that is anchored in the inner
membrane of the pathogen and projects through the outer bacterial membrane
and the host membrane (Galan & Wolf-Watz, 2006). Within it, the needle
contains a narrow channel allowing a direct delivery of the virulence factors from
the bacterial cytoplasm into the host cytoplasm. In addition to the structural
proteins that make up the needle, each type Ill secretion system also encodes for
the transcriptional regulators, virulence factors (effectors and translocators), and
chaperones (Parsot et all 2003). The virulence factors are delivered into the host
cell cytosol and interfere with the host signaling (effectors) or make a pore in the
host cell membrane (translocators) (Feldman & Cornelis, 2003). Many effectors
and translocators interact with the chaperones, which stabilize them, prevent
their aggregation, target them to the secretion apparatus, and may establish an
order of secretion for the effectors (Feldman & Cornelis, 2003, Parsot et al,
2003). These chaperones are small, acidic, homodimeric proteins that
specifically recognize only one or two effectors (Galan & Wolf-Watz, 2006).
Although there is no sequence homology between the type Ill secretion
chaperones, their three dimensional structures are remarkably similar. Each
chaperone monomer consists of an antiparallel P-sheet composed of 5 P-strands,
and 3 a-helices, all located on the same side of the P-sheet. The helix a2 and
strand P4 interact with the symmetric elements in the other monomer, forming the
patch )d 1
zk. patch 2
Figure 1.7 The structures of the Type Ill secretion system chaperones in complex with their effector substrates.
Left panes show cartoon diagrams, with the chaperones in brown and the substrates in green. Right panes show the chaperones as surface coloured according to electrostatic potential, and the substrates as cartoon in green. A) SycE in complex with the chaperone binding domain of YopE ('IL2W, (Birtalan et al, 2002)). The hydrophobic patches 1 and 2 that interact with the substrates (Birtalan) are identified by arrows. B) SicP in complex with the chaperone binding domain of SptP (IJYO, (Stebbins & Galan, 2001)). C) The heterodirneric chaperone SycNNscB in complex with a nearly full length effector YopN. SycN is in brown, YscB is in red, and YopN is in green (IXKP, (Schubot et al, 2005)).
dimer interface (Figure 1.7) (Birtalan et al, 2002, Luo et al, 2001, Stebbins &
Galan, 2001, Yip et al, 2005).
Most effectors and translocators contain a secretion signal located within
the first 20-30 residues at the N-terminus, which appears unstructured and not
conserved in sequence. The stretch of -50-100 amino acids downstream of the
signal sequence typically participates in the interaction with the chaperone and is
called a chaperone binding domain (CBD) (Galan & Wolf-Watz, 2006). There are
several crystal structures of the chaperones bound to their effectors, which
highlight the basis of their interactions. From the crystal structure of the
chaperone SycE complexed with the CBD of its substrate effector YopE from
Yersenia pseudotuberculosis, it is clear that the CBD of YopE wraps around the
dimeric SycE (Figure 1.7A) (Birtalan et all 2002). In doing so, YopE forms
extensive surface contacts with both monomers of SycE and drapes around
more than half of the circumference of the chaperone. Most of the bound CBD
from YopE is in an unfolded, extended conformation, which makes contacts with
the surface of the chaperone through main chain hydrogen bonds and numerous
specific polar and non-polar interactions. Birtalan et a1 identifies two pairs of
symmetric hydrophobic patches in SycE that interact with the secondary
structural elements found in the CBD of YopE. Patch 1 makes contact with YopE
helices a1 and a2, whereas patch 2 interacts with the strands P I and P2 (Figure
1.7A) (Birtalan et all 2002). The comparison of the SycE structures with and
without the substrate bound indicate that the chaperone structure is static and is
not affected by the substrate binding (Birtalan et all 2002). The co-crystal
structure of the chaperone SicP and the effector SptP reveals a striking similarity
to the SycE-YopE complex. SptP wraps around its chaperone in essentially the
same way as YopE, although there is no sequence similarity between either the
chaperones or the effectors (Figure 1.7B) (Stebbins & Galan, 2001).
Even though the structures described above demonstrate that the
chaperone binding domain of the effector binds to its cognate chaperone in an
unfolded form, several studies indicate that the chaperone-bound effectors still
retain their activity. It was therefore concluded that the chaperone does not
unfold the entire effector and only interacts with its CBD (Akeda & Galan, 2005,
Birtalan et al, 2002). This notion was confirmed by the co-crystal structure of the
heterodimeric chaperone SycN-YscB and its effector substrate YopN, crystallized
in nearly full length (Figure 1.7C) (Schubot et all 2005). Apart from the CBD,
which forms extensive interactions with the chaperone similar to those previously
described for SycE-YopE and SicP-SptP complexes, the rest of the effector
forms a folded domain whose conformation does not seem to be affected by the
chaperone binding (Schubot et al, 2005). CBD is required for the subsequent
interaction of the effector with a highly conserved, membrane associated
ATPase, which unfolds the entire effector prior to its secretion through the needle
apparatus (Akeda & Galan, 2005). This suggests that the chaperone masks the
aggregation-prone CBD and delivers the effector to the peripheral ATPase, which
then displaces the chaperone and unfolds the effector (Akeda & Galan, 2005,
Letzelter et al, 2006).
1 .I 1. Signal Recognition Particle
The signal recognition particle (SRP) is one of the chaperone components
of the Sec-dependent protein translocation system and is universally conserved
28
across the three kingdoms of life (Driessen et all 2001). SRP recognizes the
hydrophobic portion of the N-terminal signal peptide or internal signal anchor
sequence on the proteins that are destined for secretion or insertion into the
membrane (Fekkes & Driessen, 1999). SRP acts co-translationally, binding the
ribosome - nascent protein complex (RNC), and delivering it to the plasma
membrane (in prokaryotes) or to the endoplasmic reticulum (in eukaryotes). SRP
efficiently competes with the trigger factor for binding nascent chains at the
ribosome when a signal sequence of sufficient hydrophobicity is synthesized
(Ullers et all 2006). Eukaryotic SRP causes an arrest in further translation by the
bound ribosome (Halic & Beckmann, 2005). At the membrane, SRP-RNC
complex interacts with the SRP receptor (SR). This interaction is mediated by
GTPases that are present in both SRP and SR and activate each other in a
reciprocal fashion, which leads to docking of the RNC to the membrane and
transfer of the bound polypeptide to the Sec translocon (Egea et al, 2005, Halic &
Beckmann, 2005).
SRP is a ribonucleoprotein, which, in mammals, consists of six proteins
(SRP54, 19, 68, 72, 9, 14), and a 300-nucleotide long 7s RNA molecule. SRP
can be subdivided into two domains. The Alu domain, which is responsible for
the elongation arrest, consists of SRP9 and 14 and 3' and 5' ends of the RNA.
The S domain, necessary for signal sequence recognition and interaction with
SR, contains the remainder of the proteins and the majority of the RNA (Halic &
Beckmann, 2005). In archaea, SRP is less complex and contains only SRP54
and 19, as well as the 7s RNA similar to that found in eukaryotes. Eubacteria
il Finger loor,
I,,
Finger h
Figure 1.8 The structures of the Signal Recognition Particle.
A) A cartoon diagram of Thermus aquaticus Ffh with a peptide from a symmetry related molecule in the substrate binding groove. The NG domain is in olive, the M- domain is in red, and the peptide is in green (2FFH, (Keenan et al, 1998)) 6) Same as in A), except Ffh is shown as surface coloured according to electrostatic potential. C) A cartoon diagram of the superposition of the M-domains from structures with (2FFH) and without (lQZX, (Rosendal et al, 2003)) the bound peptide. Note the variation in position of the finger loop. D) A complex between the ribosome (green surface), eukaryotic SRP (purple cartoon) and a signal sequence peptide (red cartoon) PDB ID 2J37 (Halic et al, 2006).
contain an even simpler version of SRP, with only one protein Ffh, a homolog of
SRP54, and a 110-nucleotide long 4.5s RNA (Egea et al, 2005, Luirink &
Sinning, 2004).
In the structure of the E.coli SRP in complex with the RNC, obtained by
electron microscopy, SRP contacts the ribosomal protein L23 in the absence of
the signal peptide, and forms contacts at three other sites on the ribosome in its
presence (Schaffitzel et al, 2006). This allows SRP to scan the nascent peptide
and form full contact with the ribosome once the signal sequence is found. SRP
positions itself at the peptide exit tunnel on the ribosome, but does not cover the
peptide completely, which provides the opportunity for other factors to bind
(Schaffitzel et al, 2006).
The protein SRP54 (Ffh) and the RNA helix 8 (domain IV in eubacteria)
comprise the SRP core, which is highly conserved in all species (Luirink &
Sinning, 2004). SRP54 consists of the N-terminal NG-domain, which contains the
GTPase activity and is required for the interaction with the SRP receptor at the
membrane, and a C-terminal methionine-rich M-domain, which mediates binding
to the signal peptide and interaction with the SRP RNA (Figure 1.7) (Luirink &
Sinning, 2004). The NG and M domains are joined by a flexible linker, and the
whole molecule adopts an L-shape (Figure 1.8) (Rosendal et al, 2003).
The structure of the M-domain of SRP54 is composed of several
amphipathic a-helices that form a deep hydrophobic groove, which likely serves
as a site of the signal peptide binding. The inner lining of the peptide-binding
groove is rich in methionines, a feature that is conserved in all SRP54 (Keenan et
al, 1998, Rosendal et all 2003). Since the methionine side chain is hydrophobic
and conformationally flexible due to lack of branching, it was proposed that this
feature was an adaptation to accommodate a variety of hydrophobic residues
found in signal peptides (Bernstein et al, 1989). The N-terminal helices aM1 and
aM2 are connected by a long "finger loop" that might act like a lid to the peptide
binding site (Keenan et al, 1998, Rosendal et al, 2003). In the Thermus aquaticus
structure of the M-domain, the peptide-binding groove is occupied by a portion of
a symmetry related molecule, and thus the "finger-loop" lid is stabilized in an
"open" conformation (Figure 1.8AB) (Keenan et al, 1998). In the crystal structure
of the M-domain from Sulfolobus solfataricus, there is no peptide occupying the
hydrophobic groove, and the "finger loop" lid adopts a "closed" conformation,
folding over the C-terminal helix aM5 and covering the peptide binding groove,
which presumably stabilizes and protects it from the solvent (Figure 1.8C)
(Rosendal et all 2003). This highlights the functional role of the flexibility of the
"finger loop" lid. The closing of the "finger loop" lid causes movement of the
adjacent a-helices and rearrangement of the N-terminal part of the M-domain.
The C-terminal part of the M-domain remains stable. It contains an arginine-rich
helix-turn-helix motif that is involved in binding of SRP RNA (Rosendal et all
2003).
The recently published structures of E.coli SRP-70s RNC and mammalian
SRP-80s RNC complexes obtained by cryo-EM show that the M-domain of
SRP54 is located adjacent to the ribosomal peptide exit tunnel and the
hydrophobic groove of the M-domain is occupied by the density that corresponds
to the signal sequence (Figure 1.8D) (Halic et al, 2006).
I .12. SecB and CsaA
SecB is another chaperone involved in the Sec-dependent protein
secretion, but unlike SRP, it acts post-translationally. SecB binds to newly
synthesized pre-proteins in a non-native conformation and prevents their folding
and aggregation while delivering them to SecA, a peripherally bound component
of the Sec translocon at the inner membrane. The SecA ATPase then mediates
the insertion of the pre-protein into the SecYEG translocation channel (Driessen
et all 2001, Randall & Hardy, 2002, Zhou & Xu, 2005). SecB is only found in
Gram-negative eubacteria, and its substrates are secretory and outer membrane
proteins (Baars et all 2006). SecB does not use the N-terminal signal peptide to
recognize its substrates and binds instead to a motif of nine residues of basic
and aromatic amino acids (Knoblauch et all 1999). The secretory pre-proteins
destined for export via the SecB pathway are first recognized by the trigger
factor, which prevents the pre-protein association with SRP and enables their
interaction with SecB (Beck et all 2000, Mitra et all 2006).
CsaA is another protein that has been demonstrated to act as a
chaperone in the Sec-dependent protein secretion pathway. CsaA was first
identified in Bacillus subtilis, which lacks SecB, and was shown to bind SecA and
several secreted precursors and to prevent the protein aggregation (Linde et al,
2003, Muller et all 2000a, Muller et al, 2000b). CsaA is present in certain families
of Gram-positive eubacteria, as well as in some Gram-negative eubacteria and
archaea. CsaA prefers to bind to peptide stretches with a positive net charge,
which contain hydrophobic residues flanked by basic residues. These sequences
are likely to be found in the folded core of mature proteins, but not in the signal
peptides (Linde et al, 2003).
SecB and CsaA share no sequence or structural homology. SecB is a
homotetramer, in which each monomer is composed of a 4-stranded antiparallel
P-sheet and two a-helices located on the same side of the P-sheet. The
tetrameric molecule is organized as a dimer of dimers. First, the P-strands of the
two monomers associate to form a combined eight-stranded antiparallel P-sheet,
with the a-helices beneath it. Then, the two dimers associate via their a-helices in
such a way that the a-helices are sandwiched between the two P-sheets (Dekker
et al, 2003, Xu et al, 2000). Two long hydrophobic grooves on the interface
between the two P-sheets are present on either side of SecB, and were proposed
to be the sites of the substrate binding (Figure 1.9A) (Dekker et al, 2003, Xu et al,
2000). Xu et a1 subdivides the proposed substrate binding groove into two
subsites made up of well conserved residues: a deep subsite 1, lined with
aromatic residues, and a shallow subsite 2, lined with hydrophobic residues (Xu
et al, 2000). In addition, the substrate binding groove is rimmed with negatively
charged residues (Figure 1.9B). Overall, the biochemical nature of the substrate
binding groove correlates well with the preference of SecB to bind peptides
enriched in hydrophobic and basic residues (Xu et al, 2000). A recent study
maps the binding interface between SecB and its unfolded substrate by site-
directed spin labelling and reveals that the substrate wraps around the SecB
chaperone and binds into the grooves identified by Xu et a1 (Crane et al, 2006).
In addition to these grooves, the substrate might take several possible routes
Figure 1.9 The structures of SecB and CsaA.
A) A cartoon diagram of the homotetrameric SecB ('IFX3, (Xu et al, 2000)). B) A surface representation of SecB (1FX3). Left pane: the putative substrate binding subsites 1 and 2 are coloured green and magenta, respectively. Right pane: SecB surface coloured according to vacuum surface electrostatics. C) The structure of CsaA (2NZ0, (Shapova & Paetzel, 2007)). Left pane: a cartoon diagram of the homodimeric CsaA. Right pane: a surface representation, coloured according to protein electrostatics. The location of the putative substrate binding site is indicated by an arrow.
around the chaperone forming contacts with hydrophobic, polar, and charged
residues (Crane et al, 2006).
In contrast to SecB, CsaA is a dimer, with each monomer composed of a
5-stranded P-barrel with a short capping a-helix reminiscent of an
oligonucleotide-oligosaccharide binding (OB) fold. Short N- and C-terminal
extensions from the central P-barrel form the dimer interface. There are two large
hydrophobic cavities located at the side of each P-barrel that were proposed to
act as substrate binding sites (Figure 1.9C) (Kawaguchi et al, 2001, Shapova &
Paetzel, 2007). The entrance to each cavity contains a cluster of the negative
electrostatic surface potential, which is consistent with the fact that CsaA prefers
to bind peptides with a positive net charge. The substrate might then wrap
around the surface of CsaA in the same way as was reported for SecB (Shapova
& Paetzel, 2007).
1.13. TorD
The twin-arginine (Tat) protein transport system which exists in
eubacteria, archaea, and eukaryotic chloroplasts is dedicated to the transport of
fully folded proteins across the membrane (Lee et al, 2006, Palmer et al, 2005).
Tat substrates are respiratory enzymes that contain redox-active co-factors. They
require assembly before translocation across the membrane and therefore are
incompatible with the Sec translocation system which transports its substrates in
an unfolded form. Tat substrates are synthesized with an N-terminal signal
sequence containing SRRxFLK "twin-arginine" motif. The Tat system chaperones
were proposed to bind and "mask" this signal sequence to prevent premature
36
targeting of unassembled Tat substrates to the Tat translocon (Lee et al, 2006,
Palmer et al, 2005). They also play a role in the assembly of the substrates and
insertion of the co-factors (Lee et al, 2006).
Figure 1.10 The structure of TorD.
A cartoon representation of dimeric TorD (INIC, (Tranier et al, 2003)). Subunits A and B are shown in pink and blue, respectively.
TorD is a specific chaperone for TorA molybdoenzyme, a periplasmic
triethylamine N-oxide (TMAO) reductase. The structure of dimeric TorD from
Schewanella massilia was determined at 2.4 A resolution (Tranier et al, 2003).
TorD from this bacterium forms multiple oligomeric species; the monomeric and
the dimeric form can both facilitate assembly of TorA (Tranier et al, 2003). TorD
is made up entirely of a-helices, and the two monomers exhibit domain
swapping. Each monomer contains 10 a-helices that can be subdivided into two
domains that are linked by a hinge region. The N-terminal domain of one
monomer (6 helices) interacts with the C-terminal domain of the other monomer
(4 helices) such that the entire structure of the dimer resembles a "dumbbell"
shape with two distinct "lobes" (Figure 1 . lo) (Tranier et al, 2003). This structure,
however, may represent a folding intermediate rather than the true biological fold
of TorD, as it does not appear thermodynamically stable due to exposure of
several hydrophobic residues to the solvent (Tranier et al, 2003). The authors
suggest that each "lobe" within the structure of dimeric TorD could exist as an
independent entity formed by a single TorD monomer. Instead of domain
swapping, which requires the hinge region to be in an extended conformation,
this region could form a loop, bringing the C-terminal domain back to interact with
the N-terminal domain of the same monomer (Tranier et all 2003).
Several other Tat chaperones are known, such as DmsD, NarJ, YcdY,
HyaE, and HybE. The structures of these proteins are not yet available, and
solving them would help determine the mechanism of Tat chaperone-substrate
interaction in greater detail.
I .l4. GroEL and GroES
GroEL and its co-chaperonin GroES, also known as hsp60 and hspl0,
respectively, belong to a Group I family of chaperonins, which are found in
eubacteria and in eukaryotic mitochondria and chloroplasts. GroEL is essential in
E.coli, and although it does not bind nascent proteins, it acts downstream of the
trigger factor and DnaK and assists the folding of an estimated 10% of cytosolic
proteins (Hartl & Hayer-Hartl, 2002). Approximately 85 cytosolic proteins exhibit
an obligatory dependence on the GroEL system to reach their native state.
These are large proteins with complex topologies that fold slowly and tend to
aggregate due to prolonged exposure of hydrophobic residues during folding
(Kerner et al, 2005).
GroEL is a barrel-like assembly of 14 identical 57 kDa monomers
arranged in two heptameric stacked rings (Figure 1.9). Each ring contains a 45
&wide central cavity that is separated from the cavity in the other ring. Each
monomer consists of three domains: the apical domain that binds unfolded
substrates and GroES, the equatorial domain that binds ATP and is involved in
the interactions within and between the two rings, and a flexible intermediate
domain that connects the two other domains (Figure 1.1 1A). In the assembled
GroEL structure, the two rings are arranged such that the equatorial domains of
their subunits face each other (Figure 1.11B) (Bartolucci et al, 2005, Braig et al,
1995). The interior of the apical domains is lined with hydrophobic amino acids,
which bind the exposed hydrophobic residues on the substrates (Fenton et all
1994).
GroES is a heptameric, dome-shaped ring approximately 75 A in
diameter, composed of -10 kDa subunits arranged with a 7-fold symmetry (Hunt
et al, 1996). GroES binds to the cis ring of GroEL in the presence of ATP or ADP,
acting like a lid to cover the central chamber of GroEL (Figure 1.11C). The
interaction with GroEL occurs via a "mobile loop" segment that swings away from
the p-core of each GroES subunit and binds the apical domain on the
corresponding GroEL subunit (Xu et al, 1997). The crystal structure of the apical
domain of GroEL in complex with a phage display-selected peptide revealed that
the substrate binds into a hydrophobic groove between two a-helices, which has
also been implicated of binding the GroES mobile loop (Figure 1 .I ID) (Chen &
Sigler, 1999). These substrate binding grooves from the seven subunits of a
GroEL ring face the interior of the central GroEL cavity and surround it like an
adhesive ring that likely serves to capture parts of an unfolded polypeptide (Chen
& Sigler, 1999).
Figure 1.11 The structures of GroES and GroEL.
A) and B) The structure of apo-GroEL from E.coli (IXCK (Bartolucci et al, 2005)). A) A cartoon representation of GroEL monomer. Apical domain is in blue, intermediate domain is in red, and equatorial domain is in green. B) A surface representation of the GroEL tetradecamer. Two monomers in each ring are coloured according to the colouring scheme in A). C) A cartoon representation of the complex between E.coli GroEL and GroES (IAON, (Xu et al, 1997)). GroES is in purple, the cis ring of GroEL is in orange, and the trans ring is in green. D) A complex between the apical domain of GroEL (blue) and a phage-display derived peptide (green). PDB ID 1 DKD (Chen & Sigler, 1999). Left pane: a cartoon representation. Right pane: GroEL is shown as surface coloured according to electrostatic potential.
GroEL is a functionally asymmetric molecule; only one of its rings (the cis
ring) is active at a time, but this role is alternated between the two rings in a cycle
controlled by ATP binding and hydrolysis. The binding of GroES and the
nucleotide brings about dramatic rearrangements in the cis ring (Xu et al, 1997).
First, the movement of the intermediate domain closes the nucleotide-binding
sites in the equatorial domain so that the free dissociation of ADP from the cis
ring is impeded. Second, the apical domains undergo a large rotational and
upward motion that leads to the interaction with the "mobile loop" of GroES.
These domain movements lead to a dramatic expansion of the inner chamber of
GroEL from 85,000 A3 to 175,000 A3 and bury the hydrophobic residues that
serve to bind the substrate. This brings about a change in the biochemical
properties of the cavity lining from hydrophobic to hydrophilic and leads to a
displacement of the substrate into the central cavity of GroEL (Xu et al, 1997). As
a result, GroELIGroES complex forms a "folding cage" that can accommodate a
protein of up to 60 kDa in size. This "folding cage" provides important structural
and biochemical features that may facilitate protein folding. First, the substrates
fold in isolation from the cytosolic environment, inside a spatially confined space,
and second, the conserved hydrophobic and negatively charged residues in the
inner lining of the GroEL folding chamber may serve as chemical chaperones to
initiate folding (Tang et al, 2006). Several cycles of substrate binding and release
by GroEL may be required for the polypeptide to reach its final folded state.
GroEL exhibits a high degree of allostery, with positive cooperation in ATP
binding within the subunits of the same ring, and negative allostery in the
opposite ring (Burston & Walter, 2005, Lin & Rye, 2006). The GroEL cycle starts
with binding of the polypeptide to the apical surfaces of the trans ring, followed by
the binding of ATP. This induces the dissociation of the polypeptide and GroES
from the opposite ring and permits the binding of GroES to the trans ring. The
trans ring now becomes the cis ring. The binding of ATP and GroES brings about
dramatic rearrangements in the cis ring and displaces the substrate into the
GroEL cavity where folding occurs. The folding proceeds until all of the ATP in
the cis ring is hydrolyzed to ADP, which primes the cis complex for disassembly
(Burston & Walter, 2005, Lin & Rye, 2006).
1 .I 5. Group II Chaperonins
The group II chaperonins are represented by CCT (also known as TriC or
c-cpn) found in eukaryotic cytosol, and thermosome, found in archaea. CCT acts
as a chaperone for a distinct subset of proteins including the cytoskeletal proteins
actin and tubulin, which have an obligatory dependence on CCT for folding
(Valpuesta et al, 2005). The archaeal thermosome is thought to have a broader
substrate specificity comparable to that of hsp70 in eubacteria (Leroux, 2001).
Unlike GroEL, CCT can bind to its substrates co-translationally, and relies on its
co-chaperonin prefoldin for binding to actin and tubulin (Spiess et al, 2004).
Like GroEL, CCT and the thermosome are doughnut-shaped structures
that consist of two stacked rings. In contrast to GroEL, which is composed of
identical subunits, both CCT and the thermosome are heterooligomers
(Valpuesta et al, 2005). Each CCT ring is composed of eight homologous
monomers that on average share a 30% sequence identity. The thermosome
42
rings, on the other hand, may be composed of one or two different subunits with
an eight-fold symmetry or three different subunits with a nine-fold symmetry
(Valpuesta et all 2005). The structure of the thermosome from an archaeon
Thermoplasma acidophilum has been solved to 2.6 A resolution (Ditzel et al,
1998). Each ring of the T.acidophilum thermosome consists of 8 alternating a-
and P-subunits which share a 60% sequence identity and superimpose with an
r.m.s.d. of 1.1 A (Figure 1.12) (Ditzel et all 1998). Each ring encloses an internal
space of approximately 130,000 A3, which is significantly smaller than the central
cavity of the cis ring of GroEL. The domain composition of the subunits in the
Group I and II chaperones is similar in that each has an apical substrate binding
domain, an equatorial ATP binding domain, and an intermediate hinge domain.
However, the monomers of the Group I1 chaperonins contain an additional a-
helical protrusion to the apical domain that plays the same role as the GroES co-
chaperonin of GroEL, namely, acting as a lid to the chaperonin folding chamber
(Figure 1.12) (Ditzel et al, 1998). ATP hydrolysis triggers the closure of the lid,
which confines the substrate inside the folding chamber (Meyer et al, 2003a).
In addition to actin and tubulin, CCT binds to several other proteins that
share no common features other that many of them contain tryptophan-aspartic
acid repeats and occur as oligomeric complexes (Spiess et al, 2004). The
substrate specificity of CCT is not well defined. Both polar and hydrophobic
sequences have been implicated in the recognition of actin by CCT (Hynes &
Willison, 2000, Rommelaere et all 1999). On the other hand, two hydrophobic
sequences that mediate binding to CCT have been identified in VHL (von Hippel-
Figure 1.12 The structure of the archaeal thermosome from Thermoplasma acidophilum.
This figure was created based on PDB entry 1A6D (Ditzel et al, 1998). A) A cartoon representation of the a-subunit of the thermosome. The P-subunit is assumes a virtually identical three-dimensional structure as the a-subunit ((Ditzel et al, 1998). The equatorial domain is in green, the intermediate domain is in red, and the apical domain is in blue. The lid-like loop on the apical domain is in yellow. B) Left pane: a surface representation of the therrnosome hexadecarner. One of the a-subunits is coloured according to the scheme in A). Other a-subunits are in violet, and P- subunits are in lilac. Right pane: a cartoon representation of the view in left pane, rotated 90" along the horizontal axis.
Lindau protein), another CCT substrate (Feldman et al, 2003). Taking into
consideration the diversified substrate specificity as well as the heterooligomeric
nature of CCT, it is possible that each CCT subunit recognizes a specific motif in
the substrate. Several studies support this hypothesis. For example, a complex
between a-actin and CCT, resolved by cryo-electron microscopy, revealed that
actin binds to the apical domains of two specific CCT subunits (Llorca et al,
1999). More recently, a study by Spiess et a1 demonstrated that the different
subunits of CCT recognize specific motifs in VHL, however, the substrate
recognition is somewhat redundant among the different subunits (Spiess et all
2006). In addition, the substrate binding site in CCT occurs at a site in the apical
domain that is structurally equivalent to that of GroEL, but because CCT is
composed of different subunits, each substrate binding site has a unique pattern
of hydrophobic and polar residues (Spiess et al, 2006). Clearly, the mechanism
of substrate recognition in CCT is different from that of GroEL, which is less
specific and generally prefers to bind hydrophobic sequences.
I .I 6. The ClplHspl00 family
The members of the ClpIhsp100 protein family belong to AAA+
superfamily of ATPases associated with various cellular activities. The proteins of
the Clplhsp100 family are found in prokaryotes and in eukaryotic mitochondria
and chloroplasts, where they are involved in protein degradation or
resolubilization of protein aggregates. To carry out this function, many Clp
chaperones, such as ClpA, ClpX, and HslU, associate coaxially with a protease,
ClpP or HslV, and form ClpAP, ClpXP, or HslUV chaperone-protease complexes
(Zolkiewski, 2006). The chaperone ClpB is different in that respect because it
does not associate with a protease and is not involved in protein degradation.
Instead, the role of ClpB is to disaggregate protein aggregates, a function which
requires cooperation with the Hsp701Hsp40 chaperone system. Unlike other Clp
chaperones, ClpB is also found in the cytosol of yeast and plants (Bosl et al,
2006).
All Clp family proteins contain one (in ClpX, HslU) or two (in ClpA, ClpB)
structurally conserved ATPase domains with Walker A and Walker B nucleotide
binding motifs. In addition, most proteins contain an extra domain, which can be
found at the N-terminus, such as in ClpA and ClpB, or inserted into the
nucleotide binding domain, such as the I-domain of HslU (Dougan et al, 2002).
These extra domains have been implicated in interaction with substrates and with
adaptor proteins, which modulate the interactions between Clp chaperones and
their substrates (Dougan et all 2002).
loop 1
Figure 1.13 The structures of the ClplHsplOO family chaperones ClpA and CIpB.
A) A cartoon representation of ClpA monomer (IKSF, (Guo et al, 2002)). The N- domain is in blue, the nucleotide binding domain (NBD) 1 is in yellow, and NBD2 is in green. B) A cartoon representation of ClpB monomer (IQVR, (Lee et al, 2003)). The colouring scheme is the same as in A), except the coiled-coil insertion is in orange.
The overall architecture and the mechanism of action are similar among
the Clp chaperones. They are oligomers and contain 6 identical subunits which
form a ring-shaped structure, with ATP-binding sites located at the interfaces
between SI- buni its. The hexameric ring contains a narrow channel in the centre,
through which Clp chaperones thread their substrates in order to force their
unfolding (Maurizi & Xia, 2004, Weibezahn et al, 2004, Zolkiewski, 2006). The
inner lining of the channel contains two constrictions, or diaphragms, formed by
six mobile loops, one from each monomer. These loops are tyrosine and glycine-
rich, conserved among the different Clp chaperones, and essential for substrate
binding and translocation. It has been suggested that the movement of the loops
might help in the translocation of the substrate through the central channel
(Hinnenvisch et al, 2005). Upon translocation, ClpA, ClpX, and HslU feed their
substrates directly into the central channel of their associated protease, whereas
ClpB releases its substrates in an unfolded form to allow their refolding, perhaps
with assistance of the Hsp70/Hsp40 chaperones (Zolkiewski, 2006).
Overall, the crystal structures of ClpA and ClpB monomers are similar:
each contains an a-helical N-terminal domain, involved in substrate and adaptor
protein binding, which is followed by the two nucleotide binding domains (NBD)
arranged in tandem (Figure 1.13AB). (Guo et al, 2002, Lee et al, 2003) In
contrast to ClpA, however, ClpB contains an 85 A long coiled-coil, mobile
insertion in its nucleotide binding domain 1, whose relative position and mobility
is essential for the protein disaggregation function of the chaperone (Figure
1.136) (Lee et al, 2003). These insertions are located on the outside of the
hexameric particle and give it a propeller-like shape; their precise function is not
clear.
ClpA and ClpX recognize and bind to the ssrA tag, an I I-residue peptide
added to the C-terminus of the proteins stalled at the ribosome (Keiler et al,
1996). ClpB, on the other hand, interacts with untagged aggregated proteins. It is
presently not clear how ClpB selects its substrates. It prefers to bind to peptides
enriched in aromatic and basic residues, with a stochiometry of one peptide per
ClpB hexamer, which points to the existence of a single centrally located
substrate binding site. A conserved residue, Tyr251, is located in the central pore
of ClpB and is crucial for substrate binding (Schlieker et all 2004). In addition,
two acidic residues found in the N-terminal domain have also been implicated in
interactions with the substrates (Barnett et al, 2005). These residues are located
adjacent to a hydrophobic groove, which may be the site of the substrate binding
in the N-terminal domain (Barnett et all 2005). An equivalent hydrophobic surface
is also found in the N-terminal domain of ClpA (Xia et all 2004). It has been
suggested that in the hexameric structure, the N-terminal domains would create
a funnel-like surface that concentrates the weakly bound substrates near the
central pore (Barnett et all 2005, Xia et al, 2004).
I .I 7. Conclusion
In this chapter, several different types of molecular chaperones were
discussed. Although not by any means exhaustive, this review of the molecular
chaperones highlights several different structural features that allow these
proteins to protect their substrates from improper interactions and ensure correct
folding or participation in a proper cellular process. There does not seem to be a
unifying scheme in terms of structural features of the chaperones, although
several broadly defined structural categories can be described. For example,
several chaperones, such as TF, Skp, SurA, prefoldin, and hsp70, can be
described as "clamps" because they tend to enclose their substrates in a binding
site via arm-like structures (reviewed in Stirling et all 2006). Others, such as the
type Ill secretion chaperones, and perhaps the Sec-dependent secretion
chaperones SecB and CsaA, are relatively rigid structures that bind substrates
via small grooves on their surfaces. The pili chaperones PapD and FimC provide
missing steric information to their substrates that may prevent the substrates'
misfolding and aggregation. Type I and II chaperonins, on the other hand,
employ a completely different mechanism of interaction with substrate, enclosing
it in a central chamber with a specific microenvironment that facilitates substrate
folding. The ClpIHsp100 family chaperones thread their substrates through a
central channel to force their unfolding.
Despite the difference of the mode of interaction with substrates, it
appears that most chaperones prefer to bind to hydrophobic sequences within
their substrates, which is consistent with the fact that exposing these residues
may lead to improper folding and aggregation of the substrate proteins. Some
chaperones, such as hsp70 and GroEL, have broad substrate specificities,
whereas others, such as type Ill secretion chaperones, are more specific.
Many chaperones contain auxiliary domains within their sequence or form
quaternary interactions with other proteins which carry out a supplementary
function. Examples are Trigger Factor and SurA, which contain a peptidyl-prolyl
isomerase domain, Hsp70, which associates with various co-chaperones that
present it with specific substrates or couple it to specific pathways, and ClpA and
ClpX, which associate with the protease CIpP, which degrades their substrates.
The structures of all molecular chaperones available from the Protein Data Bank
are summarized in Appendix B, table B1.
The molecular chaperones are of great interest because they are an
essential part of the cellular machinery and are involved in numerous cellular
processes. Many chaperones play an important role in disease, such as hsp90,
an anti-cancer drug target, or type Ill secretion chaperones, which contribute to
secretion of virulence factors in pathogenic bacteria. The structures of some
potentially important chaperones, such as BiP, an hsp70 homolog from ER, or
most of the Tat secretion chaperones, have not yet been determined. Although
some chaperones have been studied in great detail, there is still a lot to be
discovered about the way these fascinating molecular machines work.
CHAPTER 2. The Crystallographic Analysis of Bacillus Subtilis CsaA
The results of the work described in this chapter were published in
Shapova YA, Paetzel M. Crystallographic analysis of Bacillus subtilis CsaA. Acta
Crystallographica. Section D: Biological Crystallography. 2007 April; 63(Part
4):478-85.
2.1. Introduction
The Sec-dependent protein targeting and translocation pathway is
universally conserved across all three domains of life (Pohlschroder et al, 2005).
In eubacteria, secreted proteins are synthesized in the cytosol as precursors
carrying an amino-terminal signal peptide (Driessen et al, 2001). These
precursors are targeted to the translocation machinery at the cytosolic
membrane. In Escherichia coli, the translocation machinery (translocase)
involves a translocation channel composed of three integral membrane proteins
SecYEG, SecA, an ATPase that provides energy for the translocation process,
and several accessory proteins, such as SecD, SecF, YajC (Figure 2.1) (de
Keyzer et al, 2003, Driessen et al, 2001, Stephenson, 2005) and YidC (Yi &
Dal bey, 2005).
In bacteria, most of the protein secretion is carried out post-translationally
(Pohlschroder et al, 2005), with the homotetrameric SecB functioning as a
targeting factor that binds to the core regions of the newly synthesized proteins
51
co-translational secretion
post-translational secretion
) ribosome
'-- i c pre-pmtein slgnal sequence
/ 1
I cytoplasm
periplasm
Figure 2.1 A schematic diagram of the Sec-dependent protein secretion in Gram-negative eubacteria.
A schematic diagram of the Sec-dependent protein secretion in Gram-negative eubacteria. The proteins destined for secretion or insertion into the membrane are synthesized in the cytosol. and are targeted to the translocation machinery at the inner membrane. Targeting occurs co-translationally via the SRP-dependent pathway or post-translationally via the SecB and CsaA dependent pathways. SRP is a GTPase that binds to the pre-protein signal sequence as it emerges from the ribosome and targets the ribosome-nascent chain complex to the membrane. At the membrane, SRP interacts with its receptor FtsY in a GTP-dependent manner, which leads to the insertion of the pre-protein into the SecYEG channel Translation continues pushing the pre-protein across the translocation channel. SecDFNajC and YidC facilitate the assembly of proteins into the inner bacterial membrane. In the SecB- and CsaA-dependent pathways, the chaperones interact with pre-proteins post-translationally, binding to an exposed core of a pre-protein. SecB and CsaA then target their substrates to the cytosolic membrane, where they interact with the ATPase SecA. SecA mediates the insertion of the pre-protein into the SecYEG translocon and provides the energy for the translocation process. On the periplasrnic side, type I signal peptidase (SPasel) cleaves the signal peptide off the secreted proteins. The Sec-dependent secretion machinery in Gram-positive B.subtilis includes the same components described above for Gram-negative eubacteria, except it lacks a SecB homologue (Yamane et al, 2004). The diagram shown above may not represent all Gram-negative eubacteria, as some species contain only SecB or only CsaA.
and targets them to the SecA subunit of the translocase, while maintaining them
in an unfolded, translocation-competent state (Driessen et al, 2001).
Interestingly, certain species of eubacteria, and Gram-positive bacteria in
particular, lack SecB. The Gram-positive eubacterial species that has been
investigated the most from a protein secretion perspective is Bacillus subtilis
(Kunst et al, 1997).
The B.subtilis csaA gene was identified as being capable of suppressing
growth and secretion defects in E.coli secA and secB mutants (Muller et al,
1992). The B.subtilis CsaA (BsCsaA) protein restored the function of thermally
inactivated firefly luciferase in chaperone mutant strains of E.coli that lacked
functional GroEL, GroES, DnaK, and DnaJ (Muller et al, 2000a). In addition,
CsaA prevented the aggregation of thermally inactivated luciferase in vitro
(Muller et al, 2000a). Furthermore, it has been demonstrated that CsaA interacts
with the SecA subunit of the Sec translocase, as well as with a number of
secreted precursors, including B.subtilis prePhoB and pre-YvaY (Linde et al,
2003, Muller et al, 2000b). BsCsaA induced the translocation of prePhoB in E.
coli membrane vesicles containing translocation machinery from B.subtilis (Muller
et al, 2000b). More recently, it has been demonstrated that the levels of
expression of the csaA gene were upregulated 3.5 fold in response to severe
secretion stress in B.subtilis (Hyyrylainen et al, 2005). The above evidence
suggests that Bacillus subtilis CsaA acts as a secretion chaperone of the Sec-
dependent protein targeting and translocation pathway, possibly compensating
for the lack of SecB.
There are several Gram-negative species of eubacteria that have both
CsaA and SecB. One of these species is Thermus thermophilus. The crystal
structure of CsaA from T.thermophilus was solved to 2.0 A resolution (Kawaguchi
et al, 2001). The functional unit of CsaA is a homodimer, in which each monomer
is composed of a P-barrel domain which resembles an
oligonucleotide1oligosaccharide binding (OB) fold, as well as short N- and C-
terminal extensions from the P-barrel core, which form the dimer interface
(Kawaguchi et al, 2001). The OB-fold proteins are incredibly diverse and have
been shown to bind a variety of substrates, such as RNA, single stranded DNA,
oligosaccharides, and proteins (Arcus, 2002). There is no sequence or structural
similarity between CsaA and SecB, which functions as a dimer of dimers with
each monomer composed of 2 a helices and 4 P strands (Dekker et al, 2003, Xu
et al, 2000).
CsaA shares sequence and structural homology with TRBPI 11, a tRNA
binding protein, as well as with the C-terminal domain of methionyl-tRNA
synthetase (Met-RS). TRBPI I I binds to the outside corner of the L-shaped
tRNA, possibly via electrostatic interactions with the tRNA phosphate backbone
and thus may act as a structure-specific tRNA chaperone (Swairjo et al, 2000).
The C-terminal domain of Met-RS also possesses general tRNA binding ability
and serves as a dimerization domain in several species (Crepin et al, 2002).
Based on the structural similarity of these two proteins to CsaA, it has been
proposed that CsaA may possess a second, tRNA binding ability (Kawaguchi et
al, 2001).
Two crystal structures of CsaA from B.subtilis, a Gram positive
eubacterium, were solved at 2.0 A and 1.9 A resolution. These structures provide
a basis for the interpretation of previous biochemical characterization of BsCsaA,
as well as a comparison with the available TtCsaA structure from the Gram-
negative organism Thermus thermophilus. In addition, these structures may
provide more clues to the mode of binding of CsaA to its proposed substrates.
2.2. Materials and Methods
2.2.1. PCR and Cloning
Genomic DNA from Bacillus subtilis was purified using the standard
phenol-chloroform method (Sambrook et al, 1989). A region of B. subtilis
genomic DNA corresponding to the csaA gene was amplified by PCR using the
forward primer 5' g agc tga ata CAT ATG gca gtt att gat gac ttt gag aaa ttg gat
atc, incorporating the Ndel restriction site (in capital letters), and the reverse
primer 5' g aat gct cat GTC GAC tta tta tcc gat ttt tgt gcc gtt tgg gac agg ctg,
incorporating the Sall restriction site. Primers were designed using the annotated
B.subtilis CsaA sequence with the Swiss-Prot accession number P37584.
PCR reaction conditions were optimized to obtain the best yield of
products. PCR reactions were carried out in a 50 pL volume, including 1X PCR
Buffer that contained 1.5 mM MgCI2 (QIAGEN), 0.8 mM dNTP, 0.5 pmollpL each
of forward and reverse primers, 2.5 U of HotStar Taq DNA polymerase
(QIAGEN), and 0.5 pg of B.subtilis genomic DNA. PCR was carried out in a
MasterGradient thermocycler (Eppendorf) with the following steps: 95•‹C for 15
min, 94•‹C for 1 min, 62•‹C for 30 sec, and 72•‹C for 1 min. The last 3 steps were
repeated for 50 cycles, followed by incubation at 4•‹C.
The amplified fragments were cloned into the pCR2.1-TOP0 vector using
the TOPO TA Cloning Kit (Invitrogen). TOPO cloning strategy takes advantage of
the single adenosine overhang at 3'end of the product introduced by Taq
polymerase during PCR reaction. The vector supplied by lnvitrogen contains 3'-T
overhangs that enable direct ligation of Taq-amplified PCR products.
Topoisomerase I covalently bound to 3' phosphates of the TOPO vector
completes the ligation reaction and releases itself from the vector. The
recombinant plasmids were transformed into TOPIOF' chemically competent
E.coli cells. Ligations and transformations were performed using materials and
instructions provided by Invitrogen. The transformed cells were grown in Luria-
Bertani (LB) media supplemented with 100 pglmL ampicillin, and plasmids were
purified using QIAGEN Plasmid Miniprep kit.
The inserts containing B.subtilis csaA gene were excised from the TOPO
vector using restriction enzymes Ndel and Sall. The inserts were separated from
the plasmid DNA by agarose gel electrophoresis; all agarose gels were stained
with ethidium bromide and the bands were visualized under ultraviolet light at
320 nm. The bands of interest were excised from the gel and purified by QIAGEN
Gel Extraction kit. The purified inserts were ligated into the expression vector
PET-28a(+) (Novagen), predigested with Ndel and Sall. This plasmid features a
cleavable N-terminal hexahistidine tag, T7 lac promoter, multiple cloning sites,
and a kanamycin resistance marker. Ligation was carried out in a 10 pL volume
and contained 0.025 pmol of NdellSall digested PET-28a(+) vector, 0.075 pmol
of or B.subtilis csaA insert, 1.25 mM ATP, 1 U of T4 DNA ligase (Invitrogen), and
1X T4 ligase buffer (Invitrogen). The ligation reaction was incubated at 4•‹C
overnight. The recombinant plasmids (BsCsaAlpET28a) were transformed into
the E.coli Nova Blue chemically competent cells for storage and propagation.
The transformation procedure involved mixing 10 pL of the ligation reaction with
100 pL of Novablue cells, incubation on ice for 5 min, heat shock at 42•‹C for 30
sec, and incubation on ice for 2 min. Next, 250 pL of LB media was added to the
reaction tube and the tube was shaken horizontally at 37•‹C and 250 rpm for 1
hour. 250 pL of transformed cells were spread on LB agar plates supplemented
with 50 pglmL kanamycin. The plates were incubated at 37•‹C overnight. Several
colonies were screened for the presence of insert by growing the transformed
cells in LB media with 50 pglmL kanamycin, purifying the plasmid with QIAGEN
Plasmid Miniprep Kit, and digesting the plasmids with Ndel and Sall restriction
enzymes. Plasmids containing the inserts were sequenced at the UBC NAPS
Sequencing facility using the universal T7 promoter primer. The sequencing
results were identical to the annotated entry for B.subtilis CsaA. The purified
BsCsaAlpET-28a constructs were transformed into E.coli BL21(DE3) chemically
competent overexpression cells. These cells express T7 RNA polymerase and
serve as expression hosts for the genes of interest. The transformation reaction
was carried out as described for NovaBlue cells, except that the length of heat
shock was 45 seconds.
2.2.2. Overexpression and Purification
Several BsCsaNpET28aIBL21 (DE3) transformants were tested for protein
overexpression in a small-scale experiment. Several colonies were picked from
the transformation plates and grown in 3 mL of LB media containing 50 pg1mL
kanamycin for 5 hours at 37•‹C. The protein expression was induced by
supplementing the cultures with 0.5 mM IPTG and incubating the cells with
rotation at 250 rpm at 37•‹C overnight. For a negative control, the cultures were
split into two equal volumes prior to IPTG addition and IPTG was added only to
one-half of each culture. The cells were pelleted and lysed with addition of 30 pL
of 50 mM Tris pH 8.0, 100 mM NaCI, 4 pL of 10 mgImL lysozyme, and 4 pL of
1800 UImL DNase. The lysis reaction was incubated on ice for 2 hours and
analyzed by SDS-PAGE. All polyacrylamide gels for SDS-PAGE were prepared
according to the recipe described in Sambrook et al, 1989, and stained with
Coomassie Brilliant Blue. The transformants overexpressing BsCsaA protein
were stored as glycerol stocks at -80•‹C.
For large scale protein overexpression, a fresh LB agarlkanamycin plate
was streaked with BsCsaNpET28aIBL21 (DE3) and incubated at 37•‹C overnight.
The next day, an overnight culture was started from a single colony of
BsCsaNpET28a/BL21(DE3) in 50 mL of LBIkanamycin. The next day, the
overnight culture was diluted into 2 L of LBIkanamycin media and grown at 37•‹C
until the measurement of the cells' optical density at 600 nm (OD600) reached 0.6.
The culture was induced for protein overexpression with 0.5 mM IPTG at 25•‹C
for 16 hours.
The cells were pelleted by centrifugation at 4000 x g for 10 min. Harvested
cells were resuspended in 50 mL of 50 mM Tris-HCI pH 8.0, 0.3 M NaCI, 10 mM
imidazole and sonicated in a Sonic Dismembrator 500 (Fischer Scientific) with 6
bursts of 30% power for 20 seconds, with 30 sec cooling periods between bursts.
Cells were lysed in a cell homogenizer (Avestin EmulsiFlex-C3), and the cell
lysate was centrifuged at 31000 x g for 30 minutes. The supernatant containing
the overexpressed His-tagged CsaA was supplemented with 10 mM imidazole
and applied to a column containing 3 mL nickel-nitriloacetic acid beads (Ni-NTA,
QIAGEN) pre-equilibrated with 50 mM Tris-HCI pH 8.0, 0.3 M NaCI, 10 mM
imidazole. The beads were washed with 20 mL of 50 mM Tris-HCI pH 8.0, 0.3 M
NaCI, 30 mM imidazole. The bound BsCsaA protein was eluted in a stepwise
manner with 3 mL fractions of 50 mM Tris pH 8.0, 300 mM NaCI, 100-400 mM
imidazole. The resulting fractions were analyzed by SDS-PAGE. In order to
remove imidazole, fractions that contained BsCsaA were pooled together and
applied to a HiTrap Sephadex G-25 desalting column (Amersham Biosciences)
pre-equilibrated with 20 mM Tris pH8.0, 0.1 M NaCI. The concentration of protein
was measured by the bicinchoninic acid (BCA) protein assay (Pierce). The His-
tag was removed by adding 20 units of thrombin per 1 mg of CsaA and
incubating at room temperature for 16 hours. The cleaved BsCsaA was passed
over the Ni-NTA again, and the flow-through fraction was concentrated and
applied to a HiPrep 16/60 Sephacryl S-100 HR size exclusion column
(Amersham Biosciences) pre-equilibrated with 20 mM Tris-HCI pH 8.0, 100 mM
NaCI. Fractions containing CsaA were pooled together and concentrated to 10
mg/mL using an Amicon ultra-centrifugal filter (Millipore).
2.2.3. Crystallization and Data Collection
The initial crystallization conditions were obtained by the hanging drop
vapour diffusion method using the sparse matrix screens from Hampton
Research. All drops contained I pL of protein and 1 pL of reservoir solution and
were hanging over 1 mL of reservoir solution. The initial crystallization condition
for trigonal BsCsaA crystals was condition #42 from the Hampton Research
Crystal Screen 1, which contained 50 mM Potassium dihydrogen phosphate and
20% PEG 8000. The crystallization conditions were further refined using grid
screens based on the initial hits by varying the pH, using additives, and changing
precipitant type and concentration. The optimized reservoir condition that
produced the P3*21 crystals used for the data collection was 0.1 M potassium
dihydrogen phosphate, 12% PEG 4000. The protein solution contained 12.5
mg/mL BsCsaA in 20 mM Tris-HCI pH 8.0, 100 mM NaCI. Crystals appeared
after 2 days of incubation at room temperature.
A different crystal condition was discovered when screening BsCsaA in a
high salt buffer against the sparse matrix screens from Hampton Research. The
reservoir condition that produced the crystals in space group P4212 was
condition #30 from Crystal Screen 1, which contained 0.2 M ammonium sulfate
and 30% PEG8000. The protein solution contained 9 mg/mL BsCsaA in 20 mM
Tris pH 8.0, 1 M NaCI. The crystals appeared after 7 days of incubation at 18•‹C.
Prior to data collection, the crystals were transferred into a cryoprotectant
solution that contained the mother liquor in which 25% of water was replaced
with glycerol. The diffraction data were collected at the Simon Fraser University
Macromolecular X-ray Diffraction Data Collection Facility using a MicroMax-007
rotating anode microfocus generator operating at 40 mV and 20 mA, VariMax Cu
HF optics, X-stream 2000 cryosystem, and R-AXIS IV++ imaging plate area
detector (MSC-Rigaku). All data were collected and processed using the
Crystalclear software pack (Pflugrath, 1999). The trigonal crystals (P3*21)
diffracted to beyond 2.0 A resolution. The tetragonal crystals (P42,2) diffracted to
beyond 1.9 A resolution. Complete data sets were collected for each crystal form.
See table 1 for the data collection statistics.
2.2.4. Structure Determination and Refinement
The structures of B.subtilis CsaA were solved by molecular replacement
using the program Phaser (McCoy et all 2005) of the CCP4 suite of programs.
The coordinates of Thermus thermophilus CsaA, chain A (1GD7A) were used as
a search model. Several rounds of restrained refinement with REFMAC5
(Murshudov et al, 1997) and manual adjustment and manipulation using Coot
(Emsley & Cowtan, 2004) were used to build the BsCsaA models. CNS (Brunger
et al, 1998) was utilized as an additional tool to carry out the combined simulated
annealing, energy minimization, and B-factor refinement. The final models were
obtained by running restrained refinement in REFMACS with TLS restraints
obtained from the TLS motion determination server (Painter & Merritt, 2006). The
quality of the final models was assessed with the program PROCHECK (Morris
et al, 1992). The coordinates for BsCsaA in space groups P3221 and P4212 were
deposited into the Protein Data Bank (Berman et all 2000) with accession
numbers 2NZ0 and 2NZH, respectively.
2.2.5. Structural Analysis
Superimpositions were carried out using the program Superpose (Maiti et
al, 2004). The surfacelbinding pocket analysis was carried out by CASTp
(Binkowski et all 2003) using a 1.4 A probe radius. The mapping of the sequence
conservation onto the three-dimensional structure was performed with
CONSURF (Glaser et al, 2003). The figures were made using PyMol (DeLano,
2002). The sequence alignment analysis (Figure 2.1 0) was prepared by ClustalW
(Thompson et al, 1994) and ESPript 2.2 (Gouet et al, 2003). The protein-protein
interaction server was used to analyze the dimer interface (Jones & Thornton,
1995). The surface electrostatic analysis was performed using the vacuum
electrostatics utility in the program PyMol.
2.3. Results and Discussion
2.3.1. PCR and Cloning
[MgC121
annealing T
DNA, CIS
Figure 2.2 The optimization of the PCR amplification of B.subtilis csaA gene.
PCR reaction was carried out in several conditions with varying temperatures of the annealing step and MgCI, and DNA concentrations. The bands corresponding to BsCsaA PCR products are identified with an arrow. 1% agarose gel was used to separate the PCR products.
The B.subtilis genomic DNA sequence corresponding to the csaA gene
was successfully amplified by PCR, producing bands which correspond to the
size of the annotated csaA gene. The results of PCR optimization are illustrated
in Figure 2.2. The PCR condition that produces the most amount of product with
the least amount of non-specific amplification contained 0.5 l ~ g of B.subtilis
genomic DNA and 1.5 mM MgCI2, with the annealing step temperature of 62•‹C.
Figure 2.3 The results of cloning the PCR-amplified B.subtilis csaA gene into pCR2.1- TOP0 vector.
The plasmids from 6 colonies of E.coli TOPIOF' cells transformed with BsCsaA- Topo construct were purified, digested with restriction enzymes Ndel and Sall to liberate the insert, and separated on 1% agarose gel. Lane 1 contains 1 b.p. DNA ladder, lanes 2-7 contain the digested constructs, and lane 8 contains the mass ruler.
Cloning of PCR-amplified csaA gene into the pCR2.1-TOP0 vector was
successful as the restriction enzyme digest of the constructs liberated bands
similar in size to the csaA gene (333 b.p.) (Figure 2.3).
Figure 2.4 The results of subcloning of the csaA gene fragment into the expression vector pET28-a(+).
The plasmids from 6 colonies of E.coli NovaBlue cells transformed with BsCsaA- pET28a construct were purified, digested with restriction enzymes Ndel and Sall to liberate the insert, and separated on 1% agarose gel. Lane 1 contains 1 b.p. DNA ladder, and lanes 2-7 contain the digested constructs.
The subcloning of csaA fragments excised from BsCsaA-Topo construct
and ligated into the overexpression plasmid pET28-a(+) was successful as the
restriction enzyme digest of the constructs liberated bands similar in size to the
csaA gene (333 b.p.) (Figure 2.4).
The sequencing of the resulting BsCsaA-pET28a construct revealed a
100% match to the annotated sequence of B.subtilis csaA gene, as well as a
partial sequence corresponding to the pET28-a(+) vector.
2.3.2. Overexpression and Purification of BsCsaA
Figure 2.5 A small-scale induction of BsCsaA expression from E.coli BL21(DE3) cells transformed with the BsCsaA-pET28a construct.
In lanes indicated by (+), cells were induced for protein expression with 0.5 mM IPTG. In lanes indicated by (-), cells were treated similarly, except no IPTG was added. Cells were lysed in a lysis buffer that contained lysozyme, and the lysates were separated on a 15% SDS-PAGE and stained with Coomasssie Blue.
The small-scale induction was successful, as the cells induced with IPTG
showed a very large band on the SDS-PAGE, whereas the cells that were not
induced did not show such band (Figure 2.5). The size of the overexpressed
protein is roughly 14 kDa, similar to that of the His-tagged BsCsaA protein
s u ~ t ft wash Elutions imidazolt
std p 10 10 30 '100 200 300 400 5 0 0 ' ~ ~
20 kDa
6 kDa
Figure 2.6 Purification of BsCsaA protein by nickel affinity chromatography.
The fractions obtained by nickel affinity chromatography were analyzed for protein content on 15% SDS-PAGE (stained with Coomassie Blue). Std: broad range standard; p: pellet obtained by centrifugation of lysed cells; supt: supernatant obtained after centrifugation of lysed cells; ft: flowthrough from the Ni-NTA column. The bands corresponding to BsCsaA protein are indicated with an arrow.
After centrifuging the cell lysates, the majority of the overexpressed
BsCsaA protein remained in the soluble form (Figure 2.6). His-tagged BsCsaA
efficiently bound to Ni-NTA beads, whereas most of the contaminated proteins
eluted in the flowthrough. BsCsaA was efficiently removed from the column with
addition of increasing concentrations of imidazole. Most of BsCsaA eluted when
buffer containing 200-400 mM imidazole was added. The eluted BsCsaA protein
was sufficiently pure, with very few of contaminating proteins present (Figure
2.6).
thrombinl
MGSSHHHHHHSSGLVPRGSHMAVIDD 6His tag
Figure 2.7 Optimization of thrombin digest of BsCsaA.
BsCsaA was incubated with 5-20 units of thrombin at 4" and at room temperature overnight. The digestion products were separated on 20% SDS-PAGE (stained with Coomassie Blue). The sequence above the gel indicates thrombin cleavage site within the N-terminal hexahistidine tag. The residues in bold indicate the N-terminus of the annotated B.subtilis CsaA sequence.
RT, OIN a, a c F e
In order to improve the crystallization efforts, the N-terminal hexahistidine
(His) tag of BsCsaA was cleaved off with thrombin. Thrombin cuts specifically at
a site within the His tag indicated by an arrow (Figure 2.7). The conditions that
produced a complete cleavage of the His tag involved digestion at room
temperature overnight using 20 U of thrombin per mg or BsCsaA (Figure 2.7).
: P % z 0 5 10 15 20 5 10 15 20 thrombin, U
- e mE w o C U
4', OIN
Figure 2.8 Purification of BsCsaA by size exclusion chromatography.
A) A profile of BsCsaA elution from the HiPrep 16/60 Sephacryl S-100 HR size exclusion column (Amersham Biosciences). The flow rate was 1 mL/min; 4 mL fractions were collected. B) The fractions corresponding to BsCsaA peak were collected, concentrated, and analyzed on a 20% SDS-PAGE stained with Coomassie Blue.
The digested BsCsaA protein was further purified by size exclusion
chromatography. BsCsaA eluted from the size exclusion column as a single
peak, indicating that no substantial amounts of contaminating proteins were
present (Figure 2.8). Fractions 37-41 were collected, combined, concentrated,
and used in subsequent crystallization experiments.
Crystallization and Data Collection
P 322 I P3221 0.05 M ammonium formate, 0.1 M potassium dihydrogen 20% PEG 8000 phosphate, 12% PEG 4000
P42,2 0.1 M ammonium sulfate, 30% PEG 8000
Figure 2.9 Crystals of B.subtilis CsaA
Space groups and reservoir conditions are indicated below the picture of each crystal.
Several different reservoir conditions produced crystals of sufficient size
and quality for X-ray data collection experiments (Figure 2.9). BsCsaA was
successfully crystallized in two space groups: P3221 and P4212. The fact that the
crystals formed in two different space groups is most likely due to different salt
concentrations in buffers that the proteins were kept in prior to setting up the
crystal drops. For crystallization in space group P3221, the protein was kept in 20
mM Tris pH 8.0, 0.1 M NaCI. For crystallization in space group P42,2, a high salt
buffer was used (20 mM Tris pH 8.0, 1 M NaCI).
The crystals shown in middle and right panes of Figure 2.9 were used for
X-ray data collection and subsequent structure determination. Trigonal crystals
(P3221) diffracted to beyond 2.0 A resolution; tetragonal crystals (P4212)
diffracted to beyond 1.9 A resolution. The data collection statistics are
summarized in Table 2.1.
Table 2.1 The crystallographic data collection statistics for B.subtilis CsaA.
DATA COLLECTION Space group P322 1 P 4 2 , 2 Unit cell dimensions (A) 148.4 x 148.4 x 54.1 109.2 x 109.2 x 37.4 Resolution range (A) 28.05 - 2.00 (2.07 - 54.57 - 1.90 (1.97 -
2.00) 1.90) Total number of reflections 209396 190906 Number of unique reflections 45675 17190 Average redundancy 4.58 (4.25) 1 1.1 1 (9.99) Oh completeness
# 98.6 (97.0) 93.3 (87.0)
Rrnerge 0.044 (0.310) 0.051 (0.286) Ilol 20.4 (4.6) 31.9 (7.2)
Values in parentheses are for the highest resolution shell. #
R m s r g e = XI1 - (I)I/X(I), where I is the observed intensity obtained from multiple observations of
symmetry-related reflections after rejections.
2.3.4. Structure Determination and Refinement
The collected diffraction data were used to solve the structures of BsCsaA
proteins. Molecular replacement using T.thermophilus CsaA structure as a
search model was utilized to solve the structures of BsCsaA. For the P3221
structure, the solution was obtained with 4 molecules in the asymmetric unit, and
for the P4212 structure, the solution was found with 2 molecules in the
asymmetric unit. The solvent content in the trigonal crystal was 65.7%, and in the
tetragonal crystal, 47.0%. Several cycles of manual adjustment with Coot and
refinement with REFMACS produced good quality models with sufficiently low R
values of 0.192 and 0.202, for the P3221 and P42,2 structures, respectively. Low
R values indicate good agreement with experimental data. Solvent atoms (water
and glycerol) were modeled in to improve agreement between the model and the
experimental results. Several residues could not be modeled due to a lack of
electron density, indicating a high degree of thermal motion. These residues
occurred at the extreme N-terminus of CsaA, and in loop 24-28. Those are most
likely flexible structures that do not assume a fixed position in the crystal. Root
mean square deviations of bonds and angles were sufficiently close to ideal
values. The Ramachandran plot analysis was utilized to assess the main-chain
conformational angles of the proteins. The main-chain angles of a vast majority
of non-glycine residues are sterically favoured, and no non-glycine residues
occur in the sterically disallowed area of the Ramachandran plot (Appendix B,
Figures B2 and B3). The summary of refinement and structure validation
statistics can be found in Table 2.2.
Table2.2 A summary of refinement statistics for the models of B.subtilis CsaA structure.
REFINEMENT P322 1 P42,2
Molecules in asymmetric unit 4 2
Number of protein atoms 3306 1654
Number of solvent atoms 320 123
Water 284 116
Glycerol 36 7
Rwork 0.192 0.202
b e e 0.230 0.245
r.m.s.d. bond lengths (A) 0.019 0.017
r.m.s.d. bond angles (") 1.78 1.61
Average B-factor (A2) - protein 17.4 17.2
Average B-factor (A2) - solvent 32.0 32.5
RAMACHANDRAN ANALYSIS (%)
Favoured 91.5 90.3
Allowed 8.5 9.1
Generous 0.0 0.6
Disallowed 0.0 0.0
Residues missing from the models Chain C: 24-28 Chain B: 1-2
due to a lack of electron density
Residues modeled as alanines due to Chain C: 32,40,91 Chain A: 1
a lack of side-chain density Chain D: 8, 22,40 Chain B: 25,27
+ R, = ZIIF~ -IF~II/ZIF~ SRf,, is calculated the same way as R factor for data omitted from refinement (5% of reflections for all data sets).
2.3.5. Sequence Alignment Analysis
h 1 s l s? h2 a3 hS B . subtilis-CsaA U - -R.QQR- eana +
W I V #AM M U HAT Iur ULT HAT 3'1s Ef S I t + LCD rzs Mt? TIE X I % RTP LID SDP YVR LYD LiC t V A i
4 a 5 sh %7 \ X + I - - - + + +
Legend t E - 3D slruchrm avrulabb - Gram-parrtlive eubactada N - Gram-nqptive eubadaria
-Archoe0 4 - inhfchain hydrogen brding through ba8bone ato& A - interchain hydrogen bonding throush rida chain at- * - backbone aiomr prrtidpate in binding site farmalion * -side chain atom patiidpate in binding site formation a . similar residues 1 -Hal rsaklrsaklm
Figure 2.10 Sequence alignment of BsCsaA and other CsaA proteins and the tRNA binding proteins MetRS and TRBPI 11.
The percent identity of each protein with BsCsaA is listed at the bottom right of each sequence. The secondary structure of BsCsaA as determined by DSSP (Kabsch & Sander, 1983) is above the alignment. The sequence numbering is that of BsCsaA. The figure below the alignment represents a stereo view of Ca trace of the BsCsaA monomer. The location of every 10th residue is indicated by a corresponding number. Portions of the polypeptide that participate in the binding site formation are shown in red.
There are three residues that appear universally conserved among the
CsaA, TRBPI 11, and MetRS proteins: Gly38, Asn69, and Ser80 (Figure 2.1 0).
Asn69 and Ser80 have been identified as residues crucial for tRNA binding in
TRBPI 11 (Swairjo et all 2000). The following residues appear to be conserved in
most CsaA proteins and are different in TRBPI 11 and MetRS: 26, 29-30, 42, 46-
51, 70, 83, 86. Based on the phylogenetic tree analysis of 18 CsaA and 18
TRBPI 11 and MetRS (C-terminal domain only) proteins (Appendix B, Figure BI),
CsaA proteins are distinct from the other group and form their own subfamily.
It is notable that the csaA gene is found in many species of Gram-positive
and Gram-negative eubacteria, as well as archaea. In the Gram-positive
eubacteria, csaA seems to be present only in the species of the genus Bacilli and
Clostridia.
2.3.6. Structural Overview
CsaA from B. subtilis (BsCsaA) is a homodimeric molecule, in which each
monomer is 110 amino acids long and has a molecular weight of 12 kDa. The
core structure of each monomer displays a well described oligonucleotide I
oligosaccharide binding (OB) fold, a 5-stranded P-barrel with a short capping a-
helix (Murzin, 1993). In the case of BsCsaA, the P-barrel is formed by strands s l ,
s2, s3, s4, s7, and an a-helix h3 located between s3 and s4 (Figure 2.1 IA). An
additional short helix h2 is found in the loop region between s2 and s3. Two short
P-strands, s5 and s6, hydrogen bond to each other and are connected by a type
II p-turn. In addition to the P-barrel, an a-helix h l is found at the N-terminus of the
protein, and the C-terminus contains strands s8 and s9. These elements
res.
Figure 2.1 1 The structure of BsCsaA.
A) A cartoon diagram of BsCsaA. Chains A and B are shown in purple and orange, respectively. (B) A Ca trace of the six superimposed chains from the two structures of BsCsaA, coloured by B-factor. Areas with the lowest B-factor are colored blue, and areas with the highest B-factor are colored red. (C) A Ca trace of the superimposed dimeric structures of BsCsaA (blue) and TtCsaA (red). Regions that show different conformations in different chains are labelled.
I participate in the formation of the dimeric structure of CsaA. I The two structures of BsCsaA differ somewhat, particularly in the positions
of atoms in residues 23-32 and 73-79 (Figure 2.116). These regions contain
residues that contribute to the formation of the putative substrate binding site.
The four chains in the asymmetric unit of the structure from the trigonal crystals
(P3221) superimpose over the backbone atoms with a root mean square
deviation (r.m.s.d.) of 0.3 A. The two chains in the asymmetric unit of the/
structure with the spacegroup P4212 superimpose with r.m.s.d. of 0.7 A. The
r.m.s.d. of superposition of all 6 chains over backbone atoms is 0.4 A.
The structural neighbours of B. subtilis CsaA were found by performing a
VAST search of the medium redundancy PDB database (Gibrat et al, 1996). The
closest structural neighbour is the CsaA protein from T thermophilus, with a
r.m.s.d. of superposition over the backbone atoms of 1.6 A (Figure 2C). Other
structural neighbours include the tRNA-binding protein TRBPI 1 1, the C-terminal
domain of methionyl-tRNA synthetase, a MetRS related protein, and the EMAPll
domain of the p43 protein from the human aminoacyl-tRNA synthetase complex. / The r.m.s.d. of superposition with these proteins and BsCsaA ranges from 2.1 A
to 3.8 A, while the sequence identity ranges from 27% to 48% (Table 2.3) - I
Notably, all of the structures described above contain tRNA binding domains.
Table 2.3 The structural neighbors of B.subtilis CsaA
1 1 PXF
Protein
CsaA
MetRS, C-
terminal
domain
TRBPI I I
EMAPll RNA
binding
domain of the
P43 Protein
TRBPI I I
Residue Rmsd*, Seq ID•˜, Organism
Range A YO
Thermus 2-109 1.57 48
thermophilus
Pyrococcus 7-112
horikoshii
Aquifex I aeolicus I
Homo 1 sapiens
Escherichia
coli 1 4-110 1 3.79
27 1 *Root mean square deviation of superposition to BsCsaA structure. 'percent sequence identity to BsCsaA.
2.3.7. The Dimerization Interface
The two monomers of BsCsaA are held together by 19 hydrogen bonds
(Table 2.4). Since the dimer has a local two-fold axis of symmetry, the same
residues form the hydrogen bonding interactions in both chains. The majority of
the inter-chain hydrogen bonding network is localized to the C-terminal portion of
the protein, and occurs through mainchain atoms (Figure 2.12C). The hydrogen
bonds that participate in dimerization are listed in Table 2.4. On average, 1550
a* (about 22.5%) of total accessible surface area is buried in the interface, which
is comprised of mostly non-polar atoms.
Figure 2.12 Dimerization of BsCsaA via hydrogen bonding interactions.
A) A fragment of the 2F,-F, electron density at 1.00, demonstrating dimerization interactions via Tyr54 hydrogen bonding to Asp100'. B) A fragment of the 2F,-F, electron density at 1.00, demonstrating dimerization interactions in the P3,21 structure via Ala2 hydrogen bonding to Asn69'. C) A ribbon diagram of BsCsaA dimer, showing the location of residues participating in dimer formation via hydrogen bonding. Residues participating in main-chain hydrogen bonds are indicated in blue. Residues participating in side-chain hydrogen bonds are shown as green sticks, and the hydrogen bonds that they form are in red.
There are two notable hydrogen bonding interactions that occur in
residues near the N-terminus. There is a hydrogen bond between the side chains
of Lys9 and Asp6' (and Lys9' and Asp6, respectively). In the P4Z12 structure, this
hydrogen bond is formed directly, from the NZ of Lys9 to the OD1 of Asp6.
However, in the P3221 structure, this hydrogen bond is indirect and occurs
through a water molecule (W151). All 4 residues (Lys9, AspG', Lys9', and Asp6)
make hydrogen bonds to water W151. The structure P3221 has two hydrogen
bonds that occur at the N-terminus, between the backbone atoms of Ala2 and
Asn69' (and vice versa) (Figure 2.12B). This interaction is not observed in the
P4Z12 structure.
Among the residues that make hydrogen bonding interactions through
their side chains, Tyr54 is notable because this residue is highly conserved
among the CsaA proteins, TRBPI 11, and the C-terminal portion of MetRS, all of
which are dimers. Tyr54 forms hydrogen bonding interactions with AsplOO
(Figure 2.12A). Although AsplOO is conserved to a lesser degree than Tyr54,
most proteins contain residues at this position capable of providing a hydrogen
bond acceptor for Tyr54. It is therefore possible that Tyr54 may be important for
the CsaA dimerization.
Table 2.4 The interchain hydrogen bonds between the two monomers of BsCsaA.
Donors Acceptors
Residue Atom Residue Atom
'Ala 2 N Asn 69' 0
#LYS 9 NZ Asp 6' OD1
Arg 53 NH1 Gln 101' OEl
Tyr 54 OH Asp 100' OD2
Lys 62 NZ Asp 100' 0 D2
Gly 86 N Gly 110' 0
Ile 88 N Lys 108' 0
Gln 98 N Gln 98' 0
• ˜ ~ l n 98 NE2 Gln 98' OEl
Asp 100 N Leu 96' 0
Gly 110 N Gly 86' 0
The inter-chain hydrogen bonds between the two monomers of BsCsaA were obtained from the optimal hydrogen bonding network (Hooft et al, 1996). These hydrogen bonds occur in both P3221 and P42,2 crystal forms. Due to a local two-fold axis of symmetry in the dimer, the same residues form hydrogen bonds in both chains of the dimer (except as noted). 'occurs only in P3,21 structure # occurs only in P42,2 structure ' occurs only once in each dimer
2.3.8. The Potential Substrate Binding Site
The CastP (Binkowski et all 2003)) analysis of the solvent accessible
surface revealed that BsCsaA contains two large T-shaped cavities, one in each
monomer. The two binding sites are separated by a rotation of approximately 90
degrees about the axis of the dimer (Figure 2.13A). These cavities are located on
one side of each P-barrel, and are formed predominantly by loops. The following
residues contribute to the binding cavity formation: 26-30, 46-52, 70-73, 75, 80-
84, 86, 88, 92, 94, 96, 110'. The cavity dimensions are approximately 15 x 15 A,
with a depth ranging from -3 to 6 A. The residues forming the cavity are mostly
Figure 2.13 The potential substrate binding sites in BsCsaA.
(A) A cartoon representation of the BsCsaA dirner with the substrate binding cavities shown as surface. Carbons are shown in green, oxygens in red, and nitrogens in blue. B) The superimposition of the residues forming the binding site from the six chains of the two structures of BsCsaA. The surface corresponds to the putative substrate binding cavity of BsCsaA structure in space group P42,2, chain A.
hydrophobic and come from the same monomer, except for the C-terminal
GlyllO. The residues that line the floor of the cavity are Ser46, Ser47, Ala48,
lle50, Ser80, Glu81, Va182, Va184, and Leu96. The three serines (Ser 46, 47, and
80) form a hydrophilic patch in the center of the cavity floor. The cavity walls are
formed by Ala26, Va128, Gln49, Phe70, Pro71, Pro72, Arg73, lle75, Leu83,
Gly86, lle88, Va194, and Glyl 10'. Most of the residues forming the binding site do
not show large variation in the position of their atoms among the six chains seen
in the two BsCsaA structures, however, the following residues show large
variation in position: Ala26, Va128, Pro29, Phe70, Pro71, Pro72, Arg73, and He75
(Figure 2.13B). These residues are located predominantly in loops that together
82
form one wall of the binding cavity. These regions demonstrated weaker electron
density and higher B-factors, which is consistent with greater mobility in this
region. These residues superimpose with a r.m.s.d. of 1.4 A (over all atoms),
whereas the r.m.s.d. for all residues of the binding site is 0.9 A, and that for all
atoms in the six models is 0.8 A. It has been previously demonstrated that CsaA
has an affinity for binding multiple peptides (Linde et al, 2003). It is possible that
the flexibility of this wall is important to accommodate the binding of a variety of
peptide substrates, which is consistent with the general chaperone function.
It has been previously shown that BsCsaA has higher affinity to denatured
peptides, thus indicating preferred binding to unfolded proteins (Linde et al,
2003). The hydrophobic nature of the binding cavity is consistent with the
chaperone activity of BsCsaA, allowing it to bind the exposed hydrophobic
residues in an unfolded protein substrate. The hydrophilic patch in the floor of the
binding cavity, formed by the three serines (Ser 46, 47, and 80), provides the
possibility for the hydrogen bonding interactions between the residues of the
cavity and the backbone atoms of the protein substrate in an extended
conformation. It is possible that the protein substrate wraps around the surface of
CsaA, much like the substrate-chaperone interactions recently described for
SecB (Crane et all 2006).
A docking experiment using the Gramm-X web server (Tovchigrechko &
Vakser, 2006) was carried out to further explore this hypothesis. Because there
is no structure of BsCsaA substrate available, a structure of the chaperone-
binding domain of YopE from a complex with a type Ill secretion chaperone
Figure 2.14 Docking of BsCsaA structure with a peptide in extended conformation.
Docking was carried out using the Gramm-X web server (Tovchigrechko & Vakser, 2006). The dimeric structure of BsCsaA in space group P3*2l, chains A and 6, was used as a receptor structure. The structure of the 55 residue long chaperone binding domain of YopE (PDB ID 1 L2W (Birtalan et al, 2002)) was used as a ligand. Prior to docking, all residues in the YopE structure (except prolines and glycines) were converted to alanines to optimize docking. The resulting docking model is shown in the figure above. BsCsaA is shown as surface, with carbons in green, oxygens in red, and nitrogens in blue. YopE is shown as sticks, with carbons in pink, oxygens in red, and nitrogens in blue. The locations of the putative substrate binding sites are identified by arrows.
SycE (PDB ID 1L2W, reviewed in section 1.9) was used as a model substrate.
This 55 residue long polypeptide is well suited for docking to BsCsaA because
most of it wraps around its chaperone SycE in an extended conformation. In
addition, the SycE chaperone is similar in size to BsCsaA. The docking model
revealed that the substrate interacts with BsCsaA in the close vicinity of both
putative substrate binding sites and wraps around the opposite side of the
chaperone in going from one substrate binding site to the other (Figure 2.14).
Due to limitations of the rigid body docking method, the conformational flexibility
of the ligand in not taken into account during docking, and therefore the substrate
is not seen to enter the potential substrate binding sites of BsCsaA in this model.
However, the model confirms that it is possible for the substrate to wrap around
BsCsaA using small grooves on its surface. In the in vitro or the in vivo situation,
it is very possible that the substrates enter both putative binding sites on BsCsaA
and may take several possible paths to wrap around the surface of the
chaperone, just as was described for SecB ((Crane et al, 2006).
Overall, the residues of the binding site are well conserved among the
sequences of CsaA proteins (Figure 2.1 0). However, some residues that are well
conserved among the CsaA proteins are different in TRBP111 and MetRS, such
as Pro29, Ser46, Gln49, Thr51. Swarjo et al identified TRBPl l I residues
important for tRNA binding (Swairjo et al, 2000). It's worth noting that most of
these residues are conserved among TRBP and CsaA, such as Ser80 (Ser82 in
TRBPIII), Arg73, and Asn69. However, two of these residues are not
conserved in CsaA proteins: Met82 (Leu83 in BsCsaA) and Glu45 (He41 in
BsCsaA). Mutating these residues in E. coli TRBP l l l reduced the binding
affinity of ~ R N A ~ ~ ~ 8-fold and 66-fold, respectively (Swairjo et al, 2000). It has
been proposed that CsaA may bind dual substrates: pre-proteins and tRNA
(Kawaguchi et all 2001). While it is possible that CsaA is capable of binding
tRNA due to its structure and sequence similarities to other tRNA-binding
proteins, the tRNA-binding ability of CsaA has not yet been demonstrated.
2.3.9. The Electrostatics and Conservation Analysis
The electrostatics analysis of the protein surface revealed two prominent
regions of electrostatic surface potential in the vicinity of the binding cavity
(Figure 2.15BC). These two areas of positive and negative electrostatic surface
potential flank the opposite sides of the binding cavity. The negative surface
potential occurs near the entrance to the binding cavity and is formed by Asp5,
Asp6, Glu8, Aspl l , and the C-terminal carboxylate (GlyllO). This negative
surface potential is consistent with proposed preference of CsaA to bind
positively charged peptides (Linde et al, 2003). An area of positive electrostatic
surface potential arises due to a cluster of basic residues at the ridge
surrounding the binding site: Arg27, Arg73, Arg74, Lys32, Lys44, and Lys79.
The electrostatics in the vicinity of the binding site differs somewhat in
BsCsaA and TtCsaA (Figure 2.15BC). The area of negative electrostatic potential
is weaker in TtCsaA than in BsCsaA and its location is shifted. This is due to the
replacement of Asp6 and Glu8 with Ala and Gln, respectively, in TtCsaA.
BsCsaA, on the other hand, contains a lysine at position 52 and a glycine at
position 90 instead of glutamic acids in TtCsaA. These replacements are
responsible for different positions of negative surface potentials in the vicinity of
the binding sites of BsCsaA and TtCsaA.
The analysis of the BsCsaA surface coloured by the conservation score
(Figure 2.15A) reveals that the following residues are highly variable: lle4', ASPS',
Glu8', Lys52, lle88, Gly90, Gln91, Asp93, GlyllO'. It is interesting to note that
these variable residues occur in a region that overlaps the area of the negative
T thermophilus
Figure 2.15 The conservation and surface electrostatic properties of BsCsaA.
A) The surface representation of BsCsaA, coloured by the conservation score, with the most conserved residues coloured dark blue, and the least conserved residues coloured red. The figure was made using ConSurf (Glaser et al, 2003). The conservation scores were obtained from sequence alignment of 36 CsaA, TRBP, and MetRS (C-terminal domains only) sequences. B and C) The protein surface electrostatics of T.thermophilus CsaA (0 ) and B.subtilis CsaA (C). Areas colored in white, red, and blue correspond to neutral, negative, and positive surface electrostatic potentials, respectively.
electrostatic surface potential at the entrance to the binding site in BsCsaA. This
region has a different pattern of electrostatic potential in TtCsaA. Based on the
high sequence variability of this region, it is possible that each CsaA protein has
its own unique pattern of electrostatic surface potential in the vicinity of the
binding site entrance, which might be optimized for the interactions with their
respective substrates.
2.4. Conclusion
CsaA is a small, dimeric protein that is present in some species of Gram-
negative and Gram-positive eubacteria and archaea. The available biochemical
data indicates that CsaA may act as a chaperone in the Sec-dependent protein
secretion system. The structure of CsaA from the Gram-positive eubacterium
B.subtilis is similar to that previously solved in the Gram-negative eubacterium T
.thermophilus. The dimeric structure is held together by 19 hydrogen bonds that
are mostly localized to the C-terminus. Seventeen of the hydrogen bonds are the
same in both P3221 and P42,2 structures, 2 hydrogen bonds are unique to each
structure. Analysis of the proposed substrate binding site reveals that it is mostly
hydrophobic with several residues forming a hydrophilic patch, which may allow
binding of unfolded peptides in an extended conformation. One wall of the
proposed binding cavity appears to be flexible, which may allow CsaA to bind a
broad spectrum of unfolded pre-protein substrates. The presence of an area of
negative surface potential near the entrance to the binding site is correlated with
the preference of CsaA to bind positively charged peptides. A region of negative
electrostatic surface potential at the entrance to the binding site in BsCsaA
contains residues that are highly variable among the sequences of CsaA,
TRBPI 11, and C-terminal regions of MetRS.
CHAPTER 3. CLONING, OVEREXPRESSION, PURIFICATION, CRYSTALLIZATION, AND REFINEMENT OF THE CRYSTAL STRUCTURES OF AGROBA CTERIUM TUMEFA CIENS CSAA
The work described in this chapter was performed in part by Dr. Anat
Feldman. My contribution to this body of work includes cloning the A.tumefaciens
CsaA construct without a peptide (AtCsaA), overexpression and purification of
the AtCsaA protein, obtaining initial crystals, as well as final refinement of the
structures of Ahmefaciens CsaA with and without a peptide.
3.1. Introduction
The components of the Sec-dependent secretion system (reviewed in
section 2.1) are similar in Gram-positive and Gram-negative eubacteria, except
for the fact that Gram-positive eubacteria lack SecB, a Sec-dependent secretion
chaperone (Yamane et al, 2004). Instead, many Gram-positive eubacteria, such
as B.subtilis, contain CsaA, another chaperone with export-related activities
(discussed in section 2.1). CsaA occurs in several Gram-positive and Gram-
negative eubacterial species, as well as in some archaea (discussed in section
2.3.5).
Agrobacterium tumefaciens is a Gram-negative eubacterium and an
important plant pathogen whose Ti plasmid is used as a valuable tool in plant
genetic engineering (Prescott et al, 2002). Unlike B.subtilis, which lacks the Sec-
dependent secretion chaperone SecB, or E.coli, which lacks CsaA,
Agrobacterium tumefaciens is one of several species that harbour both these
chaperones. CsaA protein from A.tumefaciens has 64% sequence identity to
CsaA from B.subtilis, and 48% sequence identity to CsaA from T.thermophilus.
Two crystal structures of A.tumefaciens CsaA were solved to 1.55 A and
1.65 A resolution (Feldman A. et a/, to be published). The structure (X15-AtCsaA)
that was refined to 1.65 A resolution, features a 15-residue long peptide
occupying a deep hydrophobic pocket on the surface of CsaA. The peptide was
selected from a linear peptide library displayed at the amino-terminus of phage
coat protein pVlll and showed significant binding to CsaA as determined by
ELISA. The 15-residue sequence corresponding to the selected peptide was
genetically fused to the N-terminus of CsaA prior to crystallization, using a
rationale that the N-terminus was located in close vicinity of the proposed
substrate binding site. The peptide I pocket interactions seen in the crystal
structure of XIS-AtCsaA might imitate the interactions between CsaA and its
natural pre-protein substrates. The other structure (AtCsaA), that was refined to
1.55 A resolution, was obtained from an AtCsaA construct without a peptide
tethered at the N-terminus. The two structures with and without the peptide are
compared and their pockets are analysed in detail. In addition, crystal structures
of A-tumefaciens CsaA are compared to the previously available structures of
CsaA from T.thermophilus and B.subtilis.
3.2. Materials and Methods
3.2.1. PCR and Cloning of AtCsaA and X I 5-AtCsaA
A sequence corresponding to csaA gene was amplified by PCR from
A.tumefaciens genomic DNA. The primers for PCR were designed based on a
Swiss-Prot annotated sequence with accession number Q8UDB9. PCR was
carried out with the forward primer 5' CAT ATG agc ggc gaa att tcc tat gcc gat
ttc, incorporating Ndel restriction site (in capital letters), and the reverse primer 5'
AAG CTT tca gca cat ctt ctc acc gtt cgg cac agg, incorporating Hindlll restriction
site. PCR conditions were optimized to obtain an optimal yield of products. Each
reaction was carried out in a 50 pL volume, including 1X PCR Buffer containing
1.5 mM MgCI2 (QIAGEN), 0.8 mM dNTP, 0.5 pmolIpL each of forward and
reverse primers, 2.5 U of HotStar Taq DNA polymerase (QIAGEN), and 0.5 pg of
A.tumefaciens genomic DNA. The reactions were incubated in a MasterGradient
thermocycler (Eppendorf) at 95•‹C for 15 min, then at 94•‹C for 1 min, 62•‹C for 30
sec, and 72•‹C for 1 min. The last 3 incubations were repeated for 50 cycles. The
final extension step was carried out at 72•‹C for 10 min. The amplified fragments
were cloned into the pCR2.1-TOP0 vector using the TOP0 TA Cloning Kit
(Invitrogen). The recombinant plasmids were transformed into TOP1 OF'
chemically competent E.coli cells. Ligations and transformations were performed
using materials and instructions provided by Invitrogen. The transformed cells
were grown in Luria-Bertani (LB) media supplemented with 100 pg1mL ampicillin
and plasmids were purified using QIAGEN Plasmid Miniprep kit.
The inserts containing A.fumefaciens CsaA gene were excised from the
TOP0 vector using restriction enzymes Ndel and Hindlll. The inserts were
subcloned into the PET-28a(+) overexpression vector (Novagen) designed to
express the protein with an N-terminal hexahistidine tag, and transformed into
E.coli BL21 (DE3) cells. The cloning procedure involved the same materials and
procedures as described in section 2.2.1, except that the restriction enzymes
Ndel and Hindlll were used to digest the vector. The resulting AtCsaNpET28a
construct sequenced at the UBC NAPS Sequencing facility using the universal
T7 promoter primer. The sequencing results were identical to the annotated entry
for A.fumefaciens CsaA.
The construct X15-AtCsaA was designed to express a 15-residue long
phage display derived peptide (VPGQKQHYVQPTAAN) at the N-terminus of
AtCsaA protein. For the X I 5-AtCsaA construct, primers XIS-sense: 5' gc ggc agc
CAT ATG gtt cct naq caa aas can cat tat qtt can ccn acs qca nct aat agc ggc gaa
att tc and T7-terminator 5' tat gct agt tat tgc tca g were used. Primer X15-sense
has Ndel restriction site at its 5' end (in capital letters), followed by the DNA
sequence encoding the phage display derived peptide (underlined), followed by
an overlapping region for annealing to the csaA gene. PCR was performed using
the AtCsaNpET28a construct as template. The new XIS-AtCsaA PCR product
was cloned into the vector pET28a(+) as described above, to create the plasmid
X I 5.1-CsaA-pET28.
3.2.2. Overexpression and Purification of AtCsaA and X I 5-AtCsaA
To check for AtCsaA overexpression, several BL2 1 (DE3) transformants
were grown in LB media supplemented with 50 pglmL kanamycin at 37•‹C for 4
hours, and then induced with 0.5 mM IPTG at 37•‹C for 2 hours. The pelleted
cells were lysed with addition of the lysis buffer (50 mM Tris-HCI pH8.0, 100 mM
NaCI, 40 pg/mL lysozyme, 1.8UIpL DNase) on ice for 30 min, and analyzed on
15% SDS-PAGE. All polyacrylamide gels used in SDS-PAGE were prepared
according to the recipe in Sambrook et all 1989. The protein expression was
optimized for length of induction and IPTG concentration.
For large scale protein overexpression, the same protocol as described in
section 2.2.2 was used, with the following modifications. 20 mL overnight culture
per 1 L of media were used to seed the cultures used for protein overexpression.
Cells were grown until the OD600 reading reached 0.5. The culture was induced
for protein overexpression with 0.5 mM IPTG at 37•‹C for 2 hours. The cell pellets
from each l L of culture were resuspended in 40 mL of 50 mM Tris pH 8.0, 0.3 M
NaCI. The resuspended cells were lysed in the French Pressure cell at 1000 psi
by passing through the cell 6 times. Prior to applying to Ni-NTA column, the
clarified cell supernatant was supplemented with 10 mM imidazole.
The cleavage of the His tag off AtCsaA by thrombin protease was
optimized by incubating AtCsaA with 5 and 1 units of thrombin per mg of protein
at 4•‹C and taking the aliquots at several different time points. After thrombin
cleavage, AtCsaA protein was applied to a Sephacryl S-100 HiPrep 26/60
column on an AKTA Prime system (Pharmacia Biotech), and run at 1 mL/min
93
with a buffer containing 20 mM Tris-HCI pH 8.0, 0.1 NaCI. Fractions containing
pure AtCsaA, as analyzed by SDS-PAGE, were concentrated for crystallization
using an Amicon ultra-centrifuge filter (Millipore). The protein concentration was
determined by the bicinchoninic acid (BCA) protein assay (Pierce).
For the Xl5-AtCsaA construct, a similar protocol was utilized to
overexpress, purify, and cleave the His tag off the protein, except that cells were
lysed in Avestin Emuliflex-3C cell homogenizer.
3.2.3. Crystallization and Data Collection of AtCsaA and X I 5-AtCsaA
In order to obtain crystals of AtCsaA, initial crystallization trials were
carried out using sparse matrix crystal screens (Hampton research). Initial
crystals were obtained from an aliquot of His-tagged AtCsaA by hanging drop
vapour diffusion method; the drops included 0.5 pL of 20 mg/mL AtCsaA protein
and 0.5 pL of reservoir solution and were hanging over 1 mL of reservoir solution.
Grid screens with varying pH, precipitant, and additive conditions were employed
to refine the initial crystallization conditions. To improve the quality of the
crystals, the hexahistidine tag was cleaved off the proteins with thrombin. For
subsequent experiments, sitting drop vapour diffusion at room temperature was
used to crystallize AtCsaA, and the drops consisted of 1 pl protein (13 mglml)
and 1 pl reservoir solution.
The Hampton research crystal screens were also used to obtain crystals
of X15-AtCsaA. To improve crystallization, His tag was cleaved off XIS-AtCsaA
with thrombin protease. Crystals of X15-AtCsaA were produced by the sitting
drop vapour diffusion technique at 19•‹C. Drops consisted of 1 pl protein (9
mglml) and 1 pl reservoir solution.
Prior to data collection, AtCsaA crystals were transferred into a
cryoprotectant solution that contained the mother liquor in which 15% of water
was replaced with ethylene glycol. For the crystals of X15-AtCaA, mother liquor
was used as a cryoprotectant. The diffraction data were collected at the Simon
Fraser University Macromolecular X-ray Diffraction Data Collection Facility using
a RAXlS IV++ image plate detector mounted on a 007 Rigaku X-ray generator
with VariMax CuHF optics. Data for AtCsaA and X15-AtCsaA crystals were
collected with a crystal-to-detector distance of 120 mm and 150 mm,
respectively. Data were collected at 100K using an X-stream 2000 cryo-system.
A total of 192 and 186 frames were collected for AtCsaA and X15-AtCsaA,
respectively, using 0.5O oscillations. Each image was exposed for two minutes.
Data were indexed, integrated and scaled with the program Crystal Clear
(Pflugrath, 1999).
3.2.4. Structure Determination and Refinement of AtCsaA and X I 5-AtCsaA
The structures of AtCsaA and X15-AtCsaA were solved by molecular
replacement with the program Phaserl.2 (McCoy et all 2005). The search model
used was a homology model of AtCsaA constructed with CPH (Lund et al, 2002)
based on coordinates from TtCsaA (PDB 1DG7, chain A) (Kawaguchi et all
2001). The structures were refined using restrained refinement in REFMAC5
(Murshudov et al, 1997) and simulated annealing, energy minimization and B-
factor refinement in CNS (Brunger et al, 1998). Manual adjustments to the atomic
coordinates were performed with the program Coot (Emsley & Cowtan, 2004).
The final models were obtained by running restrained refinement in REFMAC5
with TLS restraints obtained from the TLS motion determination server (Painter &
Merritt, 2006). Refinement statistics are shown in Table 3.2. The final refined
structures of AtCsaA and X15-AtCsaA atomic coordinates were deposited at the
Protein Data Bank, with accession numbers 2Q21 and 2Q2H, respectively.
3.2.5. Structural Analysis
The program PROCHECK (Morris et al, 1992) was used to analyze the
quality of the final refined model. The program Superpose (Maiti et al, 2004) was
used for superimposition of CsaA structures. The program CASTp (Binkowski et
al, 2003) was used to analyze the surface of CsaA. The hydrogen bonds were
determined with WhatlF server's optimal hydrogen bonding network (Hooft et all
1996). Intermolecular interactions were measured using the protein-protein
interaction server (Jones & Thornton, 1995). Figures were prepared with PyMol
(DeLano, 2002).
3.3. Results and Discussion
3.3.1. PCR and Cloning of AtCsaA
Figure 3.1 PCR amplification of A.tumefaciens CsaA gene.
PCR products were separated on 1% agarose gel, stained with ethidium bromide and visualized under UV light at 320nm.
PCR successfully amplified a region of genomic A.tumefaciens DNA
corresponding to the size of the annotated csaA gene. PCR worked very well, as
no non-specific products could by detected. (Figure 3.1).
CsaA - #1 #2 2
Figure 3.2 Cloning of A.tumefaciens CsaA
A) AtCsaAITopo construct purified from 2 TOPIOF' transformants and digested with restriction enzymes Ndel and Hindlll to liberate the insert. B) AtCsaAIpET28a constructs purified from 6 Novablue transformants and digested with Ndel and Hindlll to liberate the insert. Reactions were separated on a 1% agarose gel. The location of the bands corresponding to liberated A.tumefaciens csaA insert is indicated with arrows.
Cloning of PCR-amplified A.tumefaciens csaA gene into the pCR2.1-
TOP0 vector and subloning into the pET28a(+) vector was also successful. The
restriction enzyme digest of the constructs liberated bands similar in size to the
csaA gene (342 b.p.) (Figure 3.2).
3.3.2. Overexpression and Purification of AtCsaA
Figure 3.3 Purification of AtCsaA by nickel affinity chromatography.
The fractions obtained by nickel affinity chromatography were analyzed for protein content on 15% SDS-PAGE stained with Coomassie Blue. Std: broad range standard; cell supt: supernatant obtained after centrifugation of lysed cells. The arrow indicates the bands corresponding to AtCsaA protein (14.4 kDa).
The purification of His-tagged AtCsaA protein by nickel affinity
chromatography was successful because the protein bound to the Ni-NTA beads
in large quantities, whereas most of the contaminating proteins were removed in
,the flowthrough from the column (Figure 3.3). Most of AtCsaA protein eluted in
the fractions containing 200-400 mM imidazole and was sufficiently pure to carry
out initial crystallization experiments.
5U thrombin I mg AtCsaA 1 U thrombin / mg AtCsaA
1 2 4 1 6 1 1 2 4 1 6 ) . hours mubation of
Figure 3.4 Optimization of the thrombin digest reaction of A.tumefaciens CsaA.
AtCsaA protein was incubated at 4•‹C with 5 and 1 units of thrombin per mg of AtCsaA. Aliquots were taken at 1, 2, 4, and 16 hours, and reaction was quenched with addition of SDS-PAGE loading buffer. Proteins were separated on 20% SDS- PAGE and stained with Coomassie Blue.
The thrombin cleavage reaction to remove the His tag off the AtCsaA
protein was optimized using different thrombin concentration and time of
reaction. Complete cleavage was achieved when 5 units of thrombin per mg of
AtCsaA protein were added and the reaction was incubated at 4•‹C for 16 hours
(Figure 3.4).
Crystallization of AtCsaA and XIS-AtCsaA
0.1 M HEPES pH 7.5, 2% V/V PEG 400, 1.8M ammonium sulfate
0.2 M MgOAc, 0.1 M Sodium Cacodylate pH 6.5, 22% PEG 6000.
Figure 3.5 Initial crystals of AtCsaA.
A and 6) lnitial crystals produced from AtCsaA with intact His tag. C) A diffraction pattern produced by the crystal depicted in pane B.
Initial crystals of AtCsaA were obtained from Crystal Screen 1 (Hampton
Research). Methods such as varying the pH, concentration of the precipitant, and
using different additives were employed to optimize the crystallization conditions.
"Needle" crystals of AtCsaA formed in 0.1 M HEPES pH 7.3-7.7, 2% vlv
PEG 400, 1.6-2.OM ammonium sulfate (Figure 3.5A). Protein concentration was
20 mglml, in 50 mM Tris-HCI pH 8.0. First crystals appeared after 2 days of
incubation at room temperature. Removing the additive or changing it to ethanol,
glycerol, or ethylene glycol resulted in formation of bigger but more irregular
crystals.
Large crystals of a distinctive form with pointy tip, sharp facets, and flared
tails formed in 0.2 M MgOAc, 0.1 M Sodium Cacodylate pH 6.3-6.5, 20-21% wlv
PEG 8000 or 22-24% wlv PEG 6000 (Figure 3.58). Protein concentration was 20
mglml in 50 mM Tris-HCI pH 8.0, 0.1 M NaCI. First crystals appeared after 5
days of incubation at room temperature. These crystals diffracted to 2.1A at UBC
X-ray diffraction facility and to 1.5 A at the Advanced Light Source, Lawrence
Berkeley National Laboratory, University of California at Berkeley.
In order to improve AtCsaA and X15-AtCsaA crystals, thrombin protease
was used to cleave the His tag off these proteins. Crystals of AtCsaA with the His
tag cleaved off were obtained at room temperature in 1.8M ammonium sulfate,
0.1M HEPES pH 7.5, 2% PEG400, and 5% ethylene glycol. Crystals of X15-
AtCsaA with the His tag cleaved off were obtained in 30% PEG4000, 0.4M
NH~OAC, and 0.1M Na-citrate pH 5.5. These crystals were used for data
collection and structure determination. AtCsaA crystals diffracted to beyond 1.55
A resolution, whereas X15-AtCsaA crystals diffracted to beyond 1.65 A
resolution. Data collection statistics can be found in Table 3.1
Table 3.1 The data collection statistics for the structures of AtCsaA and XIS-AtCsaA.
Data Collection AtCsaA X I 5-AtCsaA
Crystallization conditions 1.8M ammonium sulfate, 30% Peg4000, 0.4M 0.1M HEPES pH 7.5, 2% NH40Ac, 0.1M Na-citrate Peg400, and 5% ethylene pH 5.5. glycol
Molecular weight (of dimer) 24,893 Da 28,246 Da Space group p61 p61 a, b, c, (A) 60.6 x 60.6 x 1 13.4 60.5 x 60.5 x 115.3 Molecules in ASU 2 2 Resolution (A) 52.53-1.55 (1.61- 1.55) 23.86 - 1.65 (1.71-1.65) Total observed reflections 3691 54 146729 Unique reflections 341 23 25681 % completeness 99.8 (99.6) 89.4 (49.7) I 1 o(l) 23.7 (7.2) 30.0 (8.6) Rrnerge (%I# 4.0 (34.1) 3.9 (16.2) Redundancy 10.8 (10.1) 5.7 (4.5)
Values in parentheses are for the highest resolution shell.
i R ~ r g e = ~ I 1 - ( l ) l I ~ ( z ) , where I is the observed intensity obtained from multiple observations of symmetry-related reflections after rejections.
3.3.4. Structure Determination and Refinement of AtCsaA and X I 5-AtCsaA
Structures of AtCsaA and X15-AtCsaA were solved by molecular
replacement, using T.thermophi1u.s CsaA structure (PDB ID lgd7) as a model.
Despite the different crystallization conditions, both AtCsaA and X15-AtCsaA
proteins produced crystals in the space group P6,, with 1 CsaA dimer in the
asymmetric unit. Interestingly, the unit cell dimensions were somewhat different,
with X15-AtCsaA unit cell being about 2 A larger at the c edge.
In order to improve the agreement between the models and the
experimental data, the datasets for AtCsaA and X15-AtCsaA were re-scaled and
averaged to resolution cut-offs of 1.55 A and 1.65 A, respectively. The models
were further improved by manual adjustment of the positions of amino acid side
chains and modeling in the solvent molecules, such as water, ethylene glycol,
citrate and sulfate ions. The refinement progress is shown in Table 3.2. The
sulfate ion was particularly important as it was found residing in the substrate
binding site in the AtCsaA structure without a peptide. (discussed in section
3.3.7). The final refinement statistics can be found in Table 3.3.
The final refined structure of AtCsaA includes all residues, except for
residues 29-31 in chain B and the N-terminal methionine in each chain, which
were not modeled due to lack of density. The residue Glu28 in chain B was
modeled as alanine due to lack of side chain density. In the structure of X15-
AtCsaA, clear electron density was obtained for the last 5 residues of the peptide
tethered at the N-terminus of molecule A (Q-6P-5T-4A3A2N-1). NO difference
density was observed for the peptide at the N-terminus of molecule B, where the
first residue seen is Gly3. Electron density was also missing for a loop region in
molecule B (residues 27-31) and the side chain of Glu43 in molecule A.
Table 3.2 The progress of refinement of AtCsaA and X15-AtCsaA structures
I 1 AtCsaA I Xl5-CsaA I
initial
final 1 0.178 1 0.208 / 0.161
Rwork
0.220
0.173
Rfree
0.245
Rwork
0.183
R h e e
0.213
Table 3.3 The refinement statistics for the structures of AtCsaA and XIS-AtCsaA.
Refinement AtCsaA X1 5-AtCsaA
Protein residues Waters Other solvent molecules
Rwork (%) Rfree (%) r.m.s. deviations Bonds (a) Angles (")
Overall B (a2) (all atoms) (protein) +I++
(peptide) (solvent)
Ramachandran "' (%)
Rwfi = ZIIFOI-IF~~~/ZIF~I SRfree is calculated the same way as R factor for data omitted from refinement (5% of reflections for all data sets). +Protein B-factor for chain A. ++ Protein B-factor for chain B. *Residues in the most favorable region **Residues in additionally allowed region
3.3.5. An Overview of the AtCsaA structure and comparison to BsCsaA
The structure of CsaA from A.tumefaciens is very similar to the structures
of CsaA from B.subtilis and T.thermophillus. It is a homodimeric molecule, in
which each monomer is 11 3 amino acids long and consists of 2 a-helices and 10
P-strands (Figure 3.6A). Strands PI, P2, P3, P4, P7, and helix a2 form the core of
the monomer, an oligonucleotide 1 oligosaccharide binding (08) fold. The N-
terminal helix a1 and the C-terminal strands P8 and P9 contain the majority of
residues that participate in formation of interchain hydrogen bonds important for
dimerization. The r.m.s.d. of superposition of dimeric AtCsaA structure and CsaA
from B.subtilis (2NZ0, chains AB) is 1.68 A over 218 a-carbons, and that of
AtCsaA and CsaA from T.thermophilus (1GD7, chains AB) is of 1.77 A over 216
a-carbons. The three structures superimpose very well except for several flexible
loop regions that deviate in position (indicated in Figure 3.6B). Like BsCsaA and
TtCsaA, AtCsaA contains two large cavities (one in each monomer) that are
separated by a rotation of approximately 90 degrees about the axis of the dimer
(Figure 3.6C). These cavities are putative substrate binding sites and are
composed of residues 26, 28-31, 33, 49-53, 73-76, 78, 83-90, 97, and residues 4'
and 11 3' from adjacent monomer. The majority of these residues are located on
the flexible loops described above, indicating that the binding site may be
dynamic. Most of the residues that make up the putative binding sites are
conserved between AtCsaA and BsCsaA. It is interesting to note that the pattern
of strong positive and negative electrostatic potential in the vicinity of the binding
site, as described for the structures of BsCsaA and TtCsaA in section 2.3.9, also
occurs in AtCsaA (Figure 3.6C). Due to high sequence variability in that region,
the position of the negatively charged area is somewhat different in all three
proteins, however, the overall tendency of having both negatively and positively
charged regions in the vicinity of the binding cavity is preserved.
res. 9
Figure 3.6 The structure of AtCsaA
A) A cartoon diagram of dimeric AtCsaA. Molecule A is in green and Molecule B is in pink. B) A ribbon diagram of the superimposed structures of AtCsaA (green), B.subtilis CsaA (2NZH, red), and T.thermophilus CsaA (1GD7, blue). C) A surface representation of the AtCsaA structure coloured according to the negative (red), positive (blue), or neutral (white) electrostatic potential. Location of the putative binding sites is indicated with arrows.
Table 3.4 The interchain hydrogen bonds between the two monomers of AtCsaA.
Donors Acceptors
Residue Atom Residue Atom
'ser 2A OG Glu 118 0
'ser 28 N Asp 14A OD2
Ile 5 N Asn 72' 0
' ~ y s 12B NZ Asp 9A OD2
Tyr 57 OH Glu 103' OEl
Asn 72 ND2 Gly 3' 0
Gly 89 N Cys 1 13' 0
Ala 101 N Ala 101' 0
Glu 103 N Leu 99' 0
' ~ r g 1048 NH1 His 56A ND1
Cys 113 N Gly 89' 0
Due to a local two-fold axis of symmetry in the dimer, the same residues form hydrogen bonds in both chains of the dimer (except as noted). Bold font indicates bonds that are conserved between AtCsaA and BsCsaA. # occurs only once per dimer
The AtCsaA dimer is held together by 18 hydrogen bonds, 14 of which are
formed by the same residues in each monomer due to the two-fold symmetry of
the dimer. The remaining 4 bonds occur only once in each dimer. The pattern of
interchain hydrogen bonding is well conserved between the structures of AtCsaA
and BsCsaA. The majority of main-chain hydrogen bonds occurs between
residues that occupy equivalent positions in sequences of AtCsaA and BsCsaA.
Two side-chain mediated hydrogen bonds are also conserved in the structures of
AtCsaA and BsCsaA and occur between the donor-acceptor pairs Tyr 57-Glu 103'
and Lysl2-Asp9'. These residues may therefore be important for dimerization of
the CsaA protein.
3.3.6. Interaction of XIS-AtCsaA with the co-crystallized peptide
The crystal structure of X15-AtCsaA reveals that the N-terminally fused
peptide from chain A of the dimer binds into a large pocket of chain A in a
symmetry related dimer. Six of the fifteen peptide residues (Q-6P-5T-A3A2N-1) in
chain A are represented by a well-defined electron density and interact with the
residues of the pocket in a symmetry related dimer. The electron density is
missing for the remainder of the peptide in chain A and for the entire peptide in
chain B. This is most likely due to a great degree of thermal motion in these
residues as they are not stabilized by interactions with other protein residues.
The peptide binds into the AtCsaA pocket in an extended conformation
and buries 446 A2 of the AtCsaA surface in the peptide-protein interface. This
surface is 58.2% non-polar in nature and consists of residues 26, 28-31, 33, 50-
52, 76, 78-80, 86, 87 from chain A and residue 113 from chain B, which contact
the peptide through both polar and non-polar interactions. There are 7 hydrogen
bonds between the atoms of the peptide and the pocket (Table 3.5). Two of
these hydrogen bonds are strictly main chain interactions and the other five are
side chain mediated. In addition to the bonds listed in Table 3.5, there is an
indirect hydrogen bond between the peptide and the pocket, which occurs via a
water molecule. The water W24 is coordinated in its position via hydrogen bonds
to the peptide atom Glen (-6) OEl and the pocket atoms Thr87A N, Ser 50A 0,
and Ser49A OG. All pocket residues that participate in hydrogen bonding
interactions do so through main chain atoms, with the exception of Arg76A. The
guanidinium group of this residue forms a bifurcated hydrogen bond to the main
Figure 3.7 The structure of AtCsaA in complex with a phage-display derived peptide (XI 5-AtCsaA).
A) A cartoon representation of the X15-AtCsaA structure. The phage-displayed peptide at the N-terminus of chain A binds into the pocket of chain A of a symmetry related dimer The two dimers are coloured green and yellow. B) A surface representation of the view in (A). Nitrogens are in blue, oxygens in red and carbons in green or yellow. C) A close-up view of the region enclosed in a box in pane (B), showing the interactions between the pocket and the peptide. Peptide residues are shown as yellow sticks, and protein residues that make contact with the peptide as green sticks. Hydrogen bonds are shown as red dashes. Peptide and protein residues are labelled in red and black, respectively.
Table 3.5 Direct hydrogen bonds between the peptide and XIS-AtCsaA.
Peptide XI 5-AtCsaA Bond
Residue Atom Residue Atom Length (A) Gln (-6) N E2 Thr 87A 0 3.1
Gln (-6) NE2 Cys 113B OXT 2.7
Gln (-6) NE2 Cys113B 0 3.4
Pro (-5) 0 Arg 76A NH1 3.0
Pro (-5) 0 Arg 76A NH2 3.2
Th r (-4) 0 Arg 30A N 2.8
Ala (-2) N Glu 28A 0 2.5
chain oxygen on the peptide residue Pro(-5).
The pocket can fit 4 peptide residues, since Asn (-1) and Ala (-2) do not
enter the pocket. Three of these residues (Gln (-6), Pro (-5), and Ala (-3)) point
down, towards the pocket and possibly act as specificity determinants for the
peptide. Gln (-6) is well coordinated in its position via three direct and one
indirect hydrogen bonds from its side chain atoms NEI and OEl to the pocket
atoms. Pro (-5) and Ala (-3) side chains interact with the pocket atoms through
non-polar contacts and are constricted in their positions by main chain hydrogen
bonds to AtCsaA. Having large residues in place of Pro(-5) and Ala (-3) in the
peptide would be disfavoured due to steric clash with the pocket atoms. Charged
residues would also likely be disfavoured due to the hydrophobic character of the
pocket at the sites of Pro(-5) and Ala (-6) binding.
Other chaperones that recognize and bind short motifs of sequence with
few specificity determinants have been characterized. For example, E.coli DnaK
chaperone was co-crystallized with a 7-residue phage display-derived peptide
which binds into a substrate binding groove in DnaK in an extended conformation
((Zhu et al, 1996), discussed in section 1.4). A leucine residue from the peptide is
buried in a deep hydrophobic pocket in DnaK and acts as a key specificity
determinant for the chaperone-peptide interaction The peptide interacts with
DnaK through side-chain mediated non-polar contacts and main-chain hydrogen
bonds. Similar chaperone-peptide interactions were described for Ydjl, an hsp40
homologue from yeast ((Li et all 2003), discussed in section 1.4). SurA, a
periplasmic chaperone, was co-crystallized with a symmetry related peptide in
the substrate binding site ((Bitto & McKay, 2002), discussed in section 1.3). In
this case, the peptide adopts an a-helical conformation and contains a leucine
and a valine residues that act as specificity determinants by interacting with the
hydrophobic pockets in the chaperone. The interactions of AtCsaA with the
peptide are similar to those described above in that AtCsaA binds to a short, 4-
residue motif in the peptide mostly through main-chain hydrogen bonding and
van der Waals interactions. Specificity likely arises from having a glutamine
residue at position (-6) in the peptide, and small uncharged residues at positions
(-5) and (-3). It is therefore possible that CsaA can bind a wide range of
substrates that display these characteristics.
3.3.7. A comparison of the substrate binding pockets in the structures of CsaA from AAumefaciens, B.subtilis, and T. thermophilus.
The position of the pocket residue Arg76 appears to be the key
determinant of the pocket's ability to bind the peptide. In molecule A, which has
the peptide bound, the guanidinium group of the Arg76 side chain points towards
the pocket, and makes a bifurcated hydrogen bond with the carbonyl oxygen of
peptide residue Pro(-5). In molecule B, which lacks the bound peptide, Arg76
points away from the pocket and towards the solvent. Interestingly, in the
structure of AtCsaA, which was crystallized without a peptide, Arg76 also points
towards the pocket in molecule A and towards the solvent in molecule B (Figure
3.8). A close examination of the pocket in molecule A in the AtCsaA structure
without a peptide revealed the presence of a sulfate ion occupying the same
position in the pocket as the peptide residue Pro(-5). Arg76 makes hydrogen
bonds to the oxygen atoms on this sulfate, similar to the interactions of Arg76
and the peptide Pro (-5) 0 in the structure with the peptide. In addition, the
sulfate ion is highly coordinated in its position via direct and indirect hydrogen
bonds to other residues of the pocket. Both the structures with and without the
peptide were crystallized in the space group P61, and with similar unit cell
dimensions. In this particular space group, steric hindrance from the symmetry
related atoms restricts the thermal motion of Arg76 from molecule A and
stabilizes it in the "down" position, which leads to narrowing of the binding pocket
and formation of stable hydrogen bonding interactions with the peptide in the
crystal. Since Arg76 sits on a solvent exposed loop, it is likely that in vitro and in
vivo, Arg76 has a much greater degree of freedom of movement, allowing it to
Pocket A
AtCsaA Pocket A
BsCsaA --
Pocket B
Pocket B
TtCsaA
Figure 3.8 The substrate binding pockets from the structures of AtCsaA, XIS-AtCsaA, BsCsaA, and TtCsaA.
Surface representations of the substrate binding pockets from the structures of X15- AtCsaA (PDB ID 2Q2H), AtCsaA (2Q21), BsCsaA (2NZO_AB), TtCsaA (1 GD7-AB). Residues important for protein-peptide interactions are shown as sticks.
move "up" and "down" and to transiently interact with and stabilize the peptide
bound into the pocket of CsaA. In the previously published structures of CsaA
from B.subtilis and T.thermophilus, an arginine and a lysine residues,
respectively, occupy positions equivalent to Arg76 in the structure of
A.tumefaciens CsaA. These residues lack a complete side chain density and are
likely flexible due to a greater degree of thermal motion. Moreover, Arg76 is
highly conserved and all CsaA proteins contain either a lysine or an arginine at
that position (Figure 2.10, alignment). This highlights the role for Arg76 as a
clamp which moves down to transiently lock and stabilize the peptide when it is
bound into the substrate binding pocket of CsaA.
The peptide atom Gln (-6) NEI makes hydrogen bonds to three main
chain atoms in the pocket (Thr87A 0, Cysl l3B OXT, and Cysl l3B 0 ) that
together make up a patch of negative charge in the floor of the peptide binding
cavity. Comparison of the substrate binding sites of CsaA structures from
A.tumefaciens, B.subtilis, and T.thermophilus reveals that the position of this
patch of negative charge is conserved among all three structures and is formed
by main chain atoms from residues that occupy equivalent positions in the
sequence (Figure 3.8). This conserved patch of negative charge in the pocket
may act as another determinant of the chaperone-peptide interaction by
providing a possibility of hydrogen bonds between the pocket and a proton donor
atom in the peptide.
3.4. Conclusion
The structure of AtCsaA in complex with a phage display-derived peptide
provides an insight into the mode of the CsaA binding to its substrates. CsaA
binds four peptide residues into an open pocket on its surface through hydrogen
bonds and van der Waals contacts. Three residues of the bound peptide might
act as specificity determinants for the chaperone-peptide interactions. Gln (-6)
forms side-chain mediated hydrogen bonds to three main chain oxygen atoms in
the pocket. These main chain oxygen atoms form a patch of negative surface
potential that is well conserved in the structures of CsaA from A.tumefaciens,
B.subtilis, and T.thermophilus, thus providing a possibility of hydrogen bonds
between the pocket and a proton donor atom in the peptide. In addition, small
uncharged residues are required at positions (-5) and (-3) of the peptide to avoid
steric clashes with the pocket atoms. The well-conserved pocket residue Arg76
acts as an important selectivity determinant for the chaperone. When the peptide
is bound, the side chain of Arg76 moves down to transiently interact with and
stabilize the peptide by forming hydrogen bonds from its guanidinium group to a
main chain oxygen on the peptide. Since AtCsaA-peptide interactions have
limited specificity, CsaA is likely to bind a wide variety of substrates thus acting
as a general chaperone.
Biochemical studies would be required in order to confirm the information
derived from the structural data on CsaA. For example, isothermal titration
calorimetry or surface plasmon resonance spectroscopy could be employed to
examine the kinetics of the phage display derived peptide binding to CsaA. In
addition, biological substrates for CsaA need to be identified in order to elucidate
its role in the Sec-dependent protein secretion.
APPENDIX A. CLONING, PURIFICATION, AND CRYSTALLIZATION OF A. TUMEFACIENS SECB
A.1. Introduction
The homotetrameric protein SecB functions as a targeting factor and a
chaperone in the Sec-dependent translocation system (discussed in section
1.12). SecB acts post-translationally, binding to the core regions of the newly
synthesized proteins and targeting them to the SecA subunit of the Sec
translocase for insertion or translocation across the membrane (Driessen et al,
2001). At the same time, SecB protects these proteins from misfolding and
aggregation. Although SecB and CsaA share no sequence or structural similarity,
they were proposed to fulfill the same function in the Sec-dependent protein
translocation and even to have an overlapping substrate specificity (Linde et all
2003). In addition, CsaA was proposed to substitute for SecB in species such as
Bacillus subtilis, which lack SecB. We chose to carry out crystallographic studies
of SecB and CsaA from A.tumefaciens simultaneously to compare the structural
features of each protein that might allow to determine the individual roles of SecB
and CsaA in Sec-dependent translocation.
A.2. Materials and Methods
A.2.1. PCR and Cloning
A sequence corresponding to secB gene was amplified by PCR from
A.tumefaciens genomic DNA. The primers for PCR reaction were designed
based on a Swiss-Prot annotated sequence with accession number Q8UJC2.
PCR was carried out with the forward primer 5' CAT ATG acc gct gaa aat ggc
gca cag ggc gca, incorporating Ndel restriction site (in capital letters), and the
reverse primer 5' AAG CTT tta gtt cgg gac agc ctg aac ctg ggc ctt, incorporating
Hindlll restriction site. PCR reaction conditions were identical to those described
for csaA from A.tumefaciens in section 2.2.1, except PCR was carried out for 43
cycles. The amplified fragments were cloned into the pCR2.1-TOP0 vector using
the TOPO TA Cloning Kit (Invitrogen). The recombinant plasmids were
transformed into TOPIOF' chemically competent E.coli cells. Ligations and
transformations were performed using materials and instructions provided by
Invitrogen. The transformed cells were grown in Luria-Bertani (LB) media
supplemented with 100 pglmL ampicillin and plasmids were purified using
QIAGEN Plasmid Miniprep kit.
The inserts containing A.tumefaciens secB gene were excised from the
TOPO vector using restriction enzymes Ndel and Hindlll, subcloned into the
PET-28a(+) overexpression vector (Novagen), and transformed into E.coli
BL21(DE3) cells using the same materials and procedures as described in
section 3.2.1. The construct AtSecBIpET28a was sequenced at the UBC NAPS
Sequencing facility using the universal T7 promoter primer. The sequencing
results were identical to the annotated entry for A.tumefaciens SecB (AtSecB).
A.2.2. Protein Overexpression and Purification
The protein expression was optimized for length of induction and IPTG
concentration using the same procedure described for AtCsaA. The conditions of
119
protein overexpression and purification by ~ i * ' affinity chromatography were
identical to those described for AtCsaA in section 3.2.2. In order to improve
crystallization conditions for AtSecB, thrombin protease was utilized to cut the
hexahistidine tag off the protein. The protein was further purified by size
exclusion chromatography using HiPrep 16/60 Sephacryl S-100 HR size
exclusion column (Amersham Biosciences) pre-equilibrated with 20 mM Tris-HCI
pH 8.0, 100 mM NaCI. Fractions containing AtSecB were pooled together and
concentrated to 10 mglmL using an Amicon ultra-centrifugal filter (Millipore).
A.2.3. Crystallization and Data Collection
The initial crystallization conditions were obtained with sparse matrix
crystal screens (Hampton Research) using hanging drop vapor diffusion method.
Drops included 0.5 pL of the reservoir solution and 0.5 pL of 10 mglml SecB and
were hanging over 1 mL reservoir solution. Initial crystals were obtained with
solution #41 from Crystal Screen I (Hampton Research), which contained 0.1M
HEPES-Na pH 7.5, 10% isopropanol, 18-20% polyethylene glycol (PEG) 8000.
Crystals were obtained after 7 days of incubation at room temperature. Grid
screens with varying pH and precipitant concentrations were employed to refine
the initial crystallization conditions. The crystallization conditions were further
optimized by varying the protein concentration, concentration and nature of the
additives, and incubation temperature. Larger crystals suitable for data collection
were obtained from an aliquot of SecB with His tag cleaved off. The reservoir
solution contained 0.2 M Mg2S04, 0.1 M Na Cacodylate pH 6.5, and 16-22%
PEG 8000. The drop contained 0.5 pL reservoir solution, 0.5 pL of 15 mglml
AtSecB protein, and 0.5 pL of 0.1% ~ l u g e n t ~ ~ detergent (Calbiochem). Crystals
appeared after two days of incubation at room temperature. An attempt to collect
data from these crystals was made at SFU Macromolecular X-ray Diffraction
Data Collection Facility using equipment and methods described in section 2.2.3.
A.3. Results and Discussion
A.3.l. PCR and Cloning
1 kb ladder SecB
ftl #3 - SecB inserts (483 b.p.)
.*
mass ruler - - 1 kbllOO ng . . , 700 bpl70 ng
500 b.pJ50 n-g
Figure A1 PCR and cloning of A.tumefaciens secB gene
A) PCR amplification of A.tumefaciens secB gene. B) AtSecBPTopo construct purified from 3 TOPIOF' transformants and digested with restriction enzymes Ndel and Hindlll to liberate the insert. C) AtSecBIpET28a constructs purified from 5 Novablue transformants and digested with Ndel and Hindlll to liberate the insert. The reaction products were separated on 1 % agarose gel.
PCR successfully amplified a region of genomic DNA from A.tumefaciens
corresponding to the annotated secB gene (Figure A1 A).The cloning of PCR-
amplified Ahmefaciens secB gene into the pCR2.1-TOP0 vector and subloning
into the pET28a(+) vector was successful as the restriction enzyme digest of the
constructs liberated bands similar in size to the secB gene (483 b.p.) (Figure A1
AB).
A.3.2. Overexpression and Purification of AtSecB protein
- - -
SecB tetramer (70 kDa)
SecB (higher MW aggregate)
Figure A2 Overexpression and purification of A.tumefaciens SecB.
A) Small scale overexpression of AtSecB. Cells were grown to an O.D. of -0.5 and induced with 0.5 mM IPTG at 37•‹C for the specified length of time. Cell lysates were separated on 15% SDS-PAGE. B) 15% SDS-PAGE of the SecB fractions obtained during purification by ~ i ' ' affinity chromatography. All gels used in SDS-PAGE were stained with Coomassie Blue. Bands corresponding to SecB are identified by arrows. C) Cleavage of the His tag off SecB by thrombin. Reactions were incubated at 4•‹C for the specified length of time and separated on 20% SDS-PAGE. D) The profile of SecB elution from the HiPrep 16160 Sephacryl S-100 HR size exclusion column (Amersharn Biosciences).
The small scale induction was successful, as the cells induced with IPTG
showed a very large band on the SDS-PAGE, which corresponds to -20 kDa,
similar to the size of a monomer of His-tagged AtSecB (Figure A2 A). The
optimal amount of AtSecB was produced after 2 hours of induction with 0.5 mM
IPTG at 37•‹C. Purification of His-tagged AtSecB by ~ i ~ ' affinity chromatography
was very efficient, as the eluted protein was very pure (Figure A2 B). Most of the
AtSecB eluted in fractions containing 200-400 mM imidazole. Thrombin cleavage
was used to remove the His tag from AtSecB. The reaction was optimized by
incubating AtSecB with 2 different concentrations of thrombin protease at 4•‹C for
1-16 hours (Figure A2 C). Cleavage was complete when 5 units of thrombin were
used per mg of AtSecB, and reaction was incubated at 4•‹C for 16 hours. In order
to separate the cleaved AtSecB from thrombin and other contaminants, an
aliquot of AtSecB was subjected to size exclusion chromatography. The size
exclusion profile showed two peaks, the larger of which corresponds to SecB
tetramer (Figure A2 D). The smaller peak corresponds to a higher molecular
weight aggregate of monomeric SecB, as confirmed by SDS-PAGE. Fractions
corresponding to tetrameric SecB were collected, concentrated, and used in
crystallization experiments.
A.3.3. Crystallization and Data Collection
A initial crystals B optimized crystals
AtSecB with His tag 0.1 M HEPES pH 7.5
10% isopropanol 18-20% PEG 8000
AtSecB without His tag 0.2 M Mg2S04
0.1 M Sodium cacodylate pH 6.5 16-22% PEG 8000
(+ 0.033% ElugentTM in the drop)
Figure A3 Crystals of A.tumefaciens SecB.
A and B) Initial and optimized crystals of AtSecB. Crystallization conditions are listed below each picture.
Initial crystals of AtSecB with His tag intact were obtained using Hampton
Research Crystal Screens. However, those crystals were too small to be used in
diffraction experiments. Optimizing crystallization conditions failed to produce
bigger crystals. In order to improve the size and quality of crystals, AtSecB with
His tag cleaved off was used in further crystallization experiments. After
extensive screening of crystallization conditions, crystals depicted in Figure A3
were obtained. These crystals looked sufficiently large and regular to be used in
diffraction experiments. However, these crystals produced no diffraction pattern.
This might be due to the fact that crystals adhered to the bottom of the sitting
drop pedestal on which they grew and were extremely difficult to remove due to
their fragility. Thus, crystals most likely were damaged during transfer to the cry0
solution. To overcome this problem, it might be necessary to use crystallization
setups other than sitting drop vapour diffusion to prevent AtSecB crystals
adhering to surfaces. Another possibility that might explain lack of diffraction from
AtSecB crystals is that the cry0 solution was not optimal and caused damage to
the crystals. If that is the case, then the cry0 solution can be optimized by
changing the nature and concentration of cryoprotectants.
APPENDIX B.
Figure 82 A sample diffraction pattern and a Ramachandran plot of the crystallographic model of BsCsaA in the space group P&21.
Figure B3 A sample diffraction pattern and a Ramachandran plot of the crystallographic model of BsCsaA in the space group P42,2.
131
Figure 64 Ramachandran plots of the crystallographic models of AtCsaA ligand-free (A) and with the symmetry related in the putative binding site (B) .
1 U9C
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earo
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hi
lus
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rmot
oga
mar
itim
a
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char
omyc
es
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ae
Xen
opus
lae
vis
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Hom
o sa
pien
s
Xen
opus
laev
is
Ho
mo
sap
iens
Bor
ek,
D.,
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l. S
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tura
l ana
lysi
s of
DJI
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erfa
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ublis
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t C
ente
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CS
G)
Cry
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e ch
aper
one
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itim
a at
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A r
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asis
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e ac
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asis
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tivity
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l . C
ell 2
006.
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95
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czak
, A
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et a
l. S
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ture
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he y
east
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H3
- A
SF
I in
tera
ctio
n: im
plic
atio
ns fo
r ch
aper
one
mec
hani
sm,
spec
ies-
spec
ific
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ract
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d ep
igen
etic
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MC
S
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t.Bio
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26
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uctu
re o
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sfl
bo
und
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e hi
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e h3
C-t
erm
inal
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ix a
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nctio
nal
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ghts
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15 p
19
l
Mou
sson
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uctu
ral b
asis
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ract
ion
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As
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ne H
3 a
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nctio
nal
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5
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2 13
2
1 CC
7
1 FE
S
1 SB
6
1 FD
8
2GG
P
1 CC
8
1 S28
1 T7S
1 l6Z
Asf
l a -
HlR
A c
ornp
lex
Atx
l
Atx
l
Atx
l
Atx
l -
Cu'
Atx
l - C
u' -
Ccc
2a
com
plex
Atx
l -
H~
''
Avr
Pph
F O
RF
2
BA
G1
(B
AG
dom
ain)
hist
one
chap
eron
e
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
met
al io
n tr
ansp
ort
prot
ein
secr
etio
n
apop
tosi
s, c
o-
chap
eron
e to
hs
p70
apop
tosi
s, c
o-
chap
eron
e to
hs
p70
X-r
ay
X-r
ay
NM
R
NM
R
NM
R,
NM
R
X-r
ay
X-r
ay
X-r
ay
NM
R
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Syn
echo
cyst
is
sp.
PC
C 6
803
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Pse
udom
onas
sy
ringa
e pv
. ph
aseo
licol
a
Cae
norh
abdi
tis
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ans
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s
Tan
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uman
AS
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A
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re o
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Arn
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Sol
utio
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he
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he y
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met
allo
chap
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e, A
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emis
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0 p1
528
Ban
ci,
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l. T
he
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l-C
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plex
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al-
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iate
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otei
n-pr
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ctio
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enzw
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stal
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re o
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e A
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met
allo
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2 A
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re
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es.
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stal
Str
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res
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ype
Ill
Eff
ecto
r P
rote
in A
vrP
phF
and
Its
Cha
pero
ne R
evea
l R
esid
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Req
uire
d fo
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norh
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tis e
lega
ns:
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re o
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omai
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ryst
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v60
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06
Bri
knar
ova,
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et a
l. S
truc
tura
l ana
lysi
s of
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G1
coch
aper
one
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its in
tera
ctio
ns w
ith H
sc70
hea
t sh
ock
prot
ein.
Nat
.Str
uct.B
io1.
200
1. v
8 p3
49
1 M62
2D9D
1 wxv
1 UK
5
1 UG
O
20
S7
1 P5U
1 Z9S
1 P5V
BA
G4I
SO
DD
(B
AG
do
mai
n)
BA
G-f
amily
mol
ecul
ar
chap
eron
e re
gula
tor-
5 (B
AG
dom
ain)
Bcl
-2 b
indi
ng
atha
noge
ne-I
(u
biqu
itin
dom
ain)
Bcl
2-as
soci
ated
at
hano
gene
3 (
BA
G
dom
ain)
Bcl
2-as
soci
ated
at
hano
gene
5 (
BA
G
dom
ain)
Caf
1 M
Ca
flM
- C
afl
- C
afl
co
mpl
ex
Ca
flM
- C
afl
- C
afl
co
mpl
ex
Ca
fl M
- C
aflc
ompl
ex
apop
tosi
s
n /a
n/a
apop
tosi
s
apop
tosi
s
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
NM
R
NM
R
NM
R
NM
R
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Ho
mo
sap
iens
Ho
mo
sap
iens
Hom
o sa
pien
s
Mus
mus
culu
s
Mus
mus
culu
s
Yer
sini
a pe
stis
Yer
sini
a pe
stis
Yer
sini
a pe
stis
Yer
sini
a pe
stis
Brik
naro
va, K
., et
al.
BA
G4l
SO
DD
pro
tein
con
tain
s a
shor
t B
AG
dom
ain.
J.B
iol.C
hem
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02.
v277
p31
172
Hat
ta,
R.,
et a
l. S
olut
ion
stru
ctur
e of
the
BA
G d
omai
n (2
75-3
50)
of B
AG
-fam
ily m
olec
ular
cha
pero
ne r
egul
ator
- 5.
To
be p
ublis
hed.
Nira
ula,
T.N
., et
al.
Sol
utio
n st
ruct
ure
of th
e ub
iqui
tin
dom
ain
of B
CL-
2 bi
ndin
g at
hano
gene
-I.
To
be
publ
ishe
d.
Hat
ta, R
., et
al.
Sol
utio
n st
ruct
ure
of th
e M
urin
e B
AG
do
mai
n of
Bcl
2-as
soci
ated
ath
anog
ene
3. T
o b
e
publ
ishe
d.
End
oh,
H.,
et a
l. S
olut
ion
stru
ctur
e of
the
firs
t M
urin
e B
AG
dom
ain
of B
cl2-
asso
ciat
ed a
than
ogen
e 5.
To
be
pu
blis
hed.
Zav
ialo
v, A
.Z.,
Kni
ght,
S.D
. A
nov
el s
elf-
capp
ing
mec
hani
sm c
ontr
ols
aggr
egat
ion
of p
erip
lasm
ic
chap
eron
e C
afl
M.
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X-r
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X-r
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U
Clp
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1 MB
V
Clp
A (
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Clp
S
com
plex
prot
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adat
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1 MB
X
Clp
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Clp
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Esc
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1 MG
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Clp
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R
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1 KH
Y
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1 UM
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1 OV
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1 YG
O
1 QU
P
2CR
L
Clp
B (
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Clp
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Clp
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Clp
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Cop
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Dna
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R
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R
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R
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R
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R
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R
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R
NM
R
X-r
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nla
nla
nla
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2.8
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heric
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Hom
o sa
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Hom
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Mus
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Hom
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Hom
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Hom
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ublis
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Bac
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2C
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1 PC
Q
1 PF
9
1 SX
4
1 AO
N
2C7D
2C7C
Gro
EL
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1 co
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A
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roE
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1 EG
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1 MN
F
1 DK
7
1 JO
N
1 LA
1
1 SR
V
1 DK
D
1 FY
9
1 FY
A
1 KID
Gro
EL
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ES
pe
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e co
mpl
ex
Gro
EL
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ptid
e co
mpl
ex
Gro
EL
(api
cal d
omai
n)
Gro
EL
(api
cal d
omai
n)
Gro
EL
(api
cal d
omai
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Gro
EL
(api
cal d
omai
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Gro
EL
(api
cal d
omai
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- dod
ecam
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pep
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Gro
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Gro
EL
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mut
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Gro
EL
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omai
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mut
ant)
prot
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prot
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ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
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X-r
ay
X-r
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X-r
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X-r
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n/a
Esc
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hia
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Esc
heric
hia
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Esc
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hia
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Esc
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hia
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The
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th
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E
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G
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L (m
utan
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mpl
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o-
EM
E
sche
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IGR
U
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ES
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EM
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roE
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haud
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ynam
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E.c
oli c
hape
roni
n G
roE
L us
ing
tran
slat
ion-
libra
tion-
sc
rew
cry
stal
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edia
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th
e E
.col
i cha
pero
nin
Gro
EL
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g tr
ansl
atio
n-lib
ratio
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scre
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ryst
allo
grap
hic
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P-A
I Fx)
, E
sche
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li X
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P
Grp
94 -
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r pr
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amin
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9O
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ound
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RP
94:
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esig
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IYT
O
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9O
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Can
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etic
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1 YT
1
1 YT
2
IYS
Z
1 QY
E
2H8M
2ES
A
2EX
L
2GQ
P
2HC
H
Grp
94 (
mut
ant)
- A
DP
co
mpl
ex
Grp
94 (
mut
ant)
- A
DP
co
mpl
ex
Grp
94 (
mut
ant)
- N
EC
A c
ompl
ex
Grp
94 (
N-t
erm
inal
do
mai
n) -
2-
chlo
rodi
deox
yade
nosi
n e
com
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Grp
94 (
N-t
erm
inal
do
mai
n) -
2-lo
do-
NE
CA
com
plex
Grp
94 (
N-t
erm
inal
do
mai
n) -
geld
anam
ycin
Grp
94 (
N-t
erm
inal
do
mai
n) -
geld
anam
ycin
com
plex
Grp
94 (
N-t
erm
inal
do
mai
n) -
ligan
d co
mpl
ex
Grp
94 (
N-t
erm
inal
do
mai
n) -
liga
nd
com
plex
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp91
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Can
is fa
rnili
aris
Can
is fa
mili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
Can
is fa
rnili
aris
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94, t
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Imm
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Imm
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Imm
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Imm
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C
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2HG
1
1 QY
5
1 UO
Y
2GF
D
1 QY
8
1 U2
0
1 uoz
1 TC
6
ITB
W
Grp
94
(N
-ter
min
al
do
ma
in)
- lig
and
com
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Grp
94
(N
-ter
min
al
do
ma
in) - N
EC
A
com
plex
Grp
94
(N
-ter
min
al
do
ma
in)
- N
-Pro
pyl
Car
boxy
amid
o A
deno
sine
com
plex
do
ma
in)
- ra
dam
ide
com
plex
Grp
94
(N
-ter
min
al
do
ma
in)
- rad
icic
ol
com
plex
Grp
94
(N
-ter
min
al
dom
ain,
mis
sing
ch
arge
d do
mai
n) -
NE
CA
com
plex
Grp
94
(N
-ter
min
al
dom
ain,
mis
sing
ch
arge
d do
mai
n) -
radi
cico
l com
plex
Grp
94
(N
-ter
min
al
dom
ain,
mut
ant)
- A
DP
Grp
94
(N
-ter
min
al
dom
ain,
mut
ant)
- A
MP
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp92
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f H
sp9O
prot
ein
fold
ing,
E
R p
aral
og o
f
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
2.3
Can
is fa
mili
aris
1.75
C
anis
fam
iliar
is
2.3
Can
is fa
mili
aris
2.3
Can
is fa
mili
aris
1.85
C
anis
fam
iliar
is
2.1
C
anis
fam
iliar
is
1.9
Can
is fa
mili
aris
1.87
C
anis
fam
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is
2.15
C
anis
fam
iliar
is
Imm
orm
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he C
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1 TC
O
1 TL5
1 FEO
I FE
E
1 TL4
1 FE
4
2JO
P
2JO
R
1 HK
9
Grp
94 (
N-t
erm
inal
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ldin
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- A
TP
E
R p
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f H
sp9O
Ha
hl
met
al io
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Ha
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2'
met
al io
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Ha
hl
- C
u'
met
al io
n tr
ansp
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Ha
hl -
Cu'
m
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ion
tran
spor
t
Ha
hl
- H
~"
m
etal
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tran
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t
hem
e tr
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hem
e tr
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regu
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tr
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RN
A
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e
X-r
ay
NM
R
X-r
ay
X-r
ay
NM
R
X-r
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X-r
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X-r
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X-r
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2.2
Can
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H
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1.75
H
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1.8
Ho
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H
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1.75
H
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1.7
Yer
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Yer
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1 ELW
1 ELR
1 S4Z
1 FP
O
1 AT
R
HO
P (
TP
RI
dom
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- H
sc70
pep
tide
com
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HO
P (
TP
R2A
dom
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-
Hsp
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eptid
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mpl
ex
HP
I (s
hado
w d
omai
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- C
AF
-1 (
PX
VX
L m
otif)
Hsc
2O (
Hsc
B)
Hsc
70 -
AD
P
1 AT
S
Hsc
70 -
AD
P
Hsc
70 (
AT
Pas
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mai
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prot
ein
fold
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ad
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hs
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hsp9
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ad
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hs
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hsp9
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mat
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asse
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a
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o-
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E
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i Hsc
A
prot
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a
cons
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hsp7
0 ho
mol
og
prot
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a
cons
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hsp7
0 ho
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prot
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a
cons
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ase
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atur
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1 HX
1 H
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X-r
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AG
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Gro
ES
-like
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mpl
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1 QQ
M
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IQQ
N
Hsc
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TP
ase
dom
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mut
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IQQ
O
Hsc
70 (A
TP
ase
dom
ain,
mut
ant)
2BU
P
Hsc
70 (A
TP
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1 XJH
117F
2Q2G
1 HD
J
1 C3G
20
37
1 NLT
IXA
O
1 HJO
1 S3X
Hsp
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N-t
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mai
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Hsp
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mai
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Hsp
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J-1,
J-
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Hsp
40
(S
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Hsp
4O (
Sis
l, J
- do
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Hsp
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ptid
e co
mpl
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C-
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ain)
Hsp
70
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TP
ase
dom
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Pas
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n) -
AD
P
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ein
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ein
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pero
ne
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ing,
co
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pero
ne
to h
sp70
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ein
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co
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pero
ne
to h
sp70
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ing,
co
-cha
pero
ne
to h
sp70
prot
ein
fold
ing,
co
-cha
pero
ne
to h
sp70
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ein
fold
ing
prot
ein
fold
ing
NM
R
X-r
ay
X-r
ay
NM
R
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Esc
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Esc
heric
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coli
Cry
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Hom
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Sac
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ae
Sac
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Sac
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. T
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19
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ase
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ain.
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03
1 BU
P
2P32
2826
1ZW
9
IZW
H
1 YE
R
IYE
S
1 US
U
Hsp
70 (
AT
Pas
e pr
otei
n fo
ldin
g do
mai
n, T
13S
mut
ant)
- A
DP
Hsp
70 (
C-t
erm
inal
10
prot
ein
fold
ing
kDa
subd
omai
n)
Hsp
7O (
Ssa
l , C
- pr
otei
n fo
ldin
g te
rmin
al d
omai
n) -
Hsp
40 (
Sis
l, C
- te
rmin
al d
omai
n)
com
plex
Hsp
82 -
inhi
bito
r pr
otei
n fo
ldin
g,
com
plex
ye
ast
hsp9
0
Hsp
82 -
rade
ster
pr
otei
n fo
ldin
g,
amin
e co
mpl
ex
yeas
t hs
p90
Hsp
9O
prot
ein
fold
ing
Hsp
9O
prot
ein
fold
ing
Hsp
9O -
Ah
al
com
plex
pr
otei
n fo
ldin
g
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
60
s ta
urus
Cae
norh
abdi
tis
eleg
ans
Dro
soph
ila
mel
anog
aste
r
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
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Hom
o sa
pien
s
Hom
o sa
pien
s
Sac
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ae
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, D
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he h
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eoni
ne 1
3 of
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ne 7
0-kD
a he
at s
hock
cog
nate
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tein
is
esse
ntia
l for
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sduc
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AT
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S
isl
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ide-
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ompl
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eren
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hen
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nd to
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GR
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plic
atio
ns f
or P
aral
og-s
peci
fic D
rug
Des
ign.
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ublis
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orm
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pt D
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GR
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plic
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aral
og-s
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rug
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1 US
V
IYE
T
2CG
9
2CG
E
1 US
7
2 F
XS
1 HK
7
2AK
P
1 AH
6
1 AH
8
Hsp
9O -
Ah
al
com
plex
Hsp
9O -
geld
anam
ycin
co
mpl
ex
Hsp
9O -
p23
(Sb
al)
co
mpl
ex
Hsp
90 -
p23
(S
ba
l)
com
plex
Hsp
9O -
p50
com
plex
Hsp
9O -
rad
amid
e co
mpl
ex
Hsp
9O (
mid
dle
dom
ain)
Hsp
9O (
mut
ant)
Hsp
9O (
N-t
erm
inal
do
mai
n)
Hsp
9O (
N-t
erm
inal
do
mai
n)
prot
ein
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ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Sac
char
omyc
es
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ae
Hom
o sa
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s
Sac
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es
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ae
Sac
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omyc
es
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Hom
o sa
pien
s
Sac
char
omyc
es
cere
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ae
Sac
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es
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Sac
char
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es
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Sac
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Sac
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1 AM
W
1 BY
Q
1AM
1
1 A4H
2BR
C
2BR
E
2CG
F
1 BG
Q
1YC
1
Hsp
9O (
N-t
erm
inal
do
mai
n) -
AD
P
Hsp
9O (
N-t
erm
inal
do
mai
n) -
AD
P -
Mg
Hsp
9O (
N-t
erm
inal
do
mai
n) -
AT
P
Hsp
9O (
N-t
erm
inal
do
mai
n) -
geld
anam
ycin
com
plex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
radi
cico
l an
alog
ue c
ompl
ex
Hsp
9O (
N-t
erm
inal
do
mai
n) -
radi
cico
l co
mpl
ex
HS
P9O
a -
dihy
drox
yphe
nylp
yraz
o le
com
plex
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Sac
char
omyc
es
cere
visi
ae
Hom
o sa
pien
s
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n an
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arac
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e H
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e H
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mol
ecul
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ryst
al S
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o B
ioch
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al
Eva
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ynth
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ryst
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itro
Bio
chem
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E
valu
atio
n of
a N
ew 3
,4-D
iary
lpyr
azol
e C
lass
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9O
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thet
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rola
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ynth
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Str
uctu
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nd B
iolo
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l E
valu
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, S.M
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asis
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n of
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H
sp9O
mol
ecul
ar c
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cico
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60
Kre
usch
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l. C
ryst
al s
truc
ture
s o
f hu
man
H
SP
9Oal
pha
com
plex
ed w
ith d
ihyd
roxy
phen
ylpy
razo
les.
B
ioor
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.Let
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15 p
1475
1YC
3
1 YC
4
2U
WD
1 UY
L
1 UY
G
IUY
I
IUY
F
HS
P9O
a -
dihy
drox
yphe
nylp
yraz
o le
com
plex
HS
P9O
a -
dihy
drox
yphe
nylp
yraz
o le
com
plex
Hsp
9Oa
- inh
ibito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n)
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
8-(
2,5-
di
met
hoxy
-ben
zyl)-
2-
fluor
o-9H
-pur
in-6
- yl
amin
e co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
8-(
2,5-
di
met
hoxy
-ben
zyl)
-2-
fluor
o-9-
pent
-9H
-pur
in-
6-yl
amin
e co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
8-(2
-chl
oro-
3,
4,5-
trim
etho
xy-
benz
yl)-
2-flu
oro-
9-
pent
-4-y
lnyl
-9H
-pur
in-
6-yl
amin
e co
mpl
ex
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Hom
o sa
pien
s
Kre
usch
, A.,
et a
l. C
ryst
al s
truc
ture
s o
f hu
man
H
SP
9Oal
pha
com
plex
ed w
ith d
ihyd
roxy
phen
ylpy
razo
les.
B
ioor
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5
Kre
usch
, A,,
et a
l. C
ryst
al s
truc
ture
s o
f hu
man
H
SP
9Oal
pha
com
plex
ed w
ith d
ihyd
roxy
phen
ylpy
razo
les.
B
ioor
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ed.C
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15
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Sha
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Inhi
bitio
n of
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Hea
t S
hock
Pro
tein
90
M
olec
ular
Cha
pero
ne in
Vitr
o a
nd
in V
ivo
by
Nov
el,
Syn
thet
ic,
Pot
ent
Res
orci
nylic
Pyr
azol
e/ls
oxaz
ole
Am
ide
Ana
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198
Wrig
ht,
L.,
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l. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng t
o H
sp9O
Iso
form
s.
Che
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iol.
2004
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l I p7
75
Wrig
ht,
L.,
et a
l. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng t
o H
sp9O
Iso
form
s.
Che
m.B
iol.
2004
. v
l I p7
75
Wrig
ht,
L., e
t al
. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng t
o H
sp9O
Iso
form
s.
Che
m.B
iol.
2004
. v
l I p7
75
Wrig
ht,
L., e
t al
. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng to
Hsp
9O Is
ofor
ms.
C
hem
.Bio
l. 20
04.
vl I p
775
1 UY
6 H
sp9O
a (N
-ter
min
al
dom
ain)
- 9
-but
yl-8
- (3
,4,5
-trim
etho
xy-
benz
yl)-
9H-p
urin
-6-
ylam
ine
com
plex
1 UY
8 H
sp9O
a (N
-ter
min
al
dom
ain)
- 9-
buty
l-8-(
3-
met
hoxy
-ben
zy1)
-9H
- pu
rin-6
-yla
min
e co
mpl
ex
1 UY
7 H
sp9O
a (N
-ter
min
al
dom
ain)
- 9
-but
yl-8
-(4-
m
etho
xy-b
enzy
l)-9H
- pu
rin-6
-yla
min
e co
mpl
ex
2BS
M
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
2BT
0 H
sp9O
a (N
-ter
min
al
dom
ain)
- in
hibi
tor
com
plex
2BY
H
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
2BY
I H
sp9O
a (N
-ter
min
al
dom
ain)
- in
hibi
tor
com
plex
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
pien
s W
right
, L.
, et
al.
Str
uctu
re-A
ctiv
ity R
elat
ions
hips
in
Pur
ine-
Bas
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hibi
tor
Bin
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to H
sp9O
Isof
orm
s.
Che
m.B
io1.
200
4. v
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75
prot
ein
fold
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X-r
ay
1.98
H
omo
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ens
Wrig
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t al
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uctu
re-A
ctiv
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elat
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hibi
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Isof
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Che
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2004
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75
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
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s W
right
, L.
, et
al. S
truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng to
Hsp
9O Is
ofor
ms.
C
hem
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75
prot
ein
fold
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X-r
ay
2.05
H
omo
sapi
ens
Dym
ock,
B.W
., et
al.
Nov
el, P
oten
t Sm
all-M
olec
ule
lnhi
bito
rs o
f the
Mol
ecul
ar C
hape
rone
Hsp
9O D
isco
vere
d T
hrou
gh S
truc
ture
-Bas
ed D
esig
n. J
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2005
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8 p4
212
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
pien
s D
ymoc
k, B
.W.,
et a
l. N
ovel
, P
oten
t Sm
all-M
olec
ule
lnhi
bito
rs o
f the
Mol
ecul
ar C
hape
rone
Hsp
9O D
isco
vere
d T
hrou
gh S
truc
ture
-Bas
ed D
esig
n. J
.Med
.Che
m.
2005
. v4
8 p4
2 12
prot
ein
fold
ing
X-r
ay
1.9
Hom
o sa
pien
s B
roug
h, P
.A.,
et a
l. 3-
(5-C
hlor
o-2,
4-D
ihyd
roxy
phen
y1)-
P
yraz
ole-
4-C
arbo
xam
ides
as
lnhi
bito
rs o
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Hsp
9O
Mol
ecul
ar C
hape
rone
. Bio
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.Che
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ett.
2005
. v15
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197
prot
ein
fold
ing
X-r
ay
1.6
Hom
o sa
pien
s B
roug
h, P
.A.,
et a
l. 3-
(5-C
hlor
o-2,
4-D
ihyd
roxy
phen
y1)-
P
yraz
ole-
4-C
arbo
xam
ides
as
lnhi
bito
rs o
f the
Hsp
9O
Mol
ecul
ar C
hape
rone
. B
ioor
g.M
ed.C
hem
.Let
t. 20
05.
v15
p519
7
2H55
2F
Wz
2FW
Y
1 UY
M
1 XQ
R
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
inhi
bito
r co
mpl
ex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
PU
-DZ
8 in
hibi
tor
com
plex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
PU
-H64
in
hibi
tor
com
plex
Hsp
9Oa
(N-t
erm
inal
do
mai
n) -
PU
-H71
in
hibi
tor
com
plex
Hsp
9OP
- 9-
buty
l- 8(
3,4,
5-tr
imet
hoxy
- be
nzyl
)-9H
-pur
in-6
- yl
amin
e
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
prot
ein
fold
ing
Hsp
BP
l (c
ore
dom
ain)
re
gula
tion
of
Hsp
70 fu
nctio
n
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
1.9
Hom
o sa
pien
s
1.79
H
omo
sapi
ens
2.3
Hom
o sa
pien
s
2.7
Hom
o sa
pien
s
1.9
Hom
o sa
pien
s
2.0
Hom
o sa
pien
s
2.1
Hom
o sa
pien
s
2.1
Hom
o sa
pien
s
2.45
H
omo
sapi
ens
2.1
Hom
o sa
pien
s
Bar
ril, X
., et
al.
Str
uctu
re-B
ased
Dis
cove
ry o
f a
New
C
lass
of H
sp9O
Inh
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rs. B
ioor
g.M
ed.C
hem
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t. 20
05.
vl5
~5
18
7
Bar
ril, X
., et
al.
4-A
min
o D
eriv
ativ
es o
f th
e H
sp9O
In
hibi
tor C
ct01
8159
. B
ioor
g.M
ed.C
hem
.Let
t. 20
06. v
16
p254
3
Bar
ril, X
., et
al.
4-A
min
o D
eriv
ativ
es o
f th
e H
sp9O
ln
hibi
tor
Cct
0181
59.
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org.
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m.L
ett.
2006
. v1
6 p2
543
Bar
ril, X
., et
al.
4-A
min
o D
er~
vativ
es of t
he H
sp9O
ln
hibi
tor
Cct
0181
59.
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org.
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ett.
2006
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p2
543
How
es, R
., et
al.
A F
luor
esce
nce
Pol
ariz
atio
n A
ssay
for
Inhi
bito
rs o
f H
sp9O
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chem
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6. v
350
p202
Imm
orm
ino,
R.M
., et
al.
Str
uctu
ral a
nd q
uant
um c
hem
ical
st
udie
s of
8-a
ryl-s
ulfa
nyl a
deni
ne c
lass
Hsp
9O in
hibi
tors
. J.
Med
.Che
m. 2
006.
v49
p49
53
Imm
orm
ino,
R.M
., et
al.
Str
uctu
ral a
nd q
uant
um c
he
m~
cal
stud
ies
of 8
-ary
l-sul
fany
l ade
nine
cla
ss H
sp9O
inhi
bito
rs.
J.M
ed.C
hem
. 200
6. v
49 p
4953
Imm
orm
ino,
R.M
., et
al.
Str
uctu
ral a
nd q
uant
um c
hem
ical
st
udie
s of
8-a
ryl-s
ulfa
nyl a
deni
ne c
lass
Hsp
9O in
hibi
tors
. J.
Med
.Che
m. 2
006.
v49
p49
53
Wrig
ht,
L., e
t al
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truc
ture
-Act
ivity
Rel
atio
nshi
ps in
P
urin
e-B
ased
lnhi
bito
r B
indi
ng to
Hsp
9O Is
ofor
ms.
C
hem
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04. v
l 1 p
775
Sho
mur
a, Y
., et
al.
Reg
ulat
ion
of H
sp70
Fun
ctio
n by
H
spB
PI;
Str
uctu
ral A
naly
sis
Rev
eals
an
Alte
rnat
e M
echa
nism
for
Hsp
70 N
ucle
otid
e E
xcha
nge.
Mol
.Cel
l 20
05. v
17 p
367
1 XQ
S
21W
S
21W
U
21W
X
1 SF
8
210Q
1 Y
4S
1 Y
4U
210P
Hsp
BP
l (c
ore
dom
ain)
re
gula
tion
of
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
X-r
ay
Ho
mo
sap
iens
S
hom
ura,
Y.,
et a
l. R
egul
atio
n of
Hsp
70 F
unct
ion
by
Hsp
BP
l; S
truc
tura
l Ana
lysi
s R
evea
ls a
n A
ltern
ate
Mec
hani
sm fo
r H
sp70
Nuc
leot
ide
Exc
hang
e. M
ol.C
ell
2005
. v1
7 p3
67
- H
sp70
(A
TP
ase
dom
ain)
com
plex
~
g~
70
fu
nctio
n
Hsp
9O (
N-t
erm
inal
do
mai
n) -
radi
cico
l pr
otei
n fo
ldin
g,
Sac
char
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es
cere
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ae
Pro
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N.,
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l. ln
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Syn
thet
ic
Mac
rola
cton
es:
Syn
thes
is a
nd S
truc
tura
l an
d B
iolo
gica
l E
valu
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Hsp
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N-t
erm
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mai
n) -
radi
cico
l pr
otei
n fo
ldin
g,
Sac
char
omyc
es
cere
visi
ae
Pro
isy,
N.,
et a
l. ln
hibi
tion
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sp9O
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Syn
thet
ic
Mac
rola
cton
es:
Syn
thes
is a
nd S
truc
tura
l and
Bio
logi
cal
Eva
luat
ion
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ing
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d C
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iona
l Ana
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N.,
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l. ln
hibi
tion
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sp9O
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Syn
thet
ic
Mac
rola
cton
es:
Syn
thes
is a
nd
Str
uctu
ral a
nd
Bio
logi
cal
Eva
luat
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Con
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atio
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Hsp
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erm
inal
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rad
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ol
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ein
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S
acch
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ce
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Htp
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E
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li hs
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ryst
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min
al D
imer
izat
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Dom
ain
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e E
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iona
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li H
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l. C
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t sh
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79
Htp
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AD
P
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ein
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E. c
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heric
hia
coli
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i, Q
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n re
arra
ngem
ent
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k pr
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n 90
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Htp
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AD
P
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l Ana
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li S
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ls D
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ase
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Htp
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al
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otei
n fo
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65
Esc
heric
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X-r
ay
Hum
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ctiv
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ase
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al
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nla
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Hum
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mai
n)
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ain
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mai
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88
5 p
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UN
R
PR
OT
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88
5
prot
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ain)
lnvB
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ipA
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R
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H
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Ill
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mpl
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urkh
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L
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2CU
G
1 ZX
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8
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R
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MB
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19
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ublis
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nucl
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U
1 EJF
2PJH
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1 QP
X
3DP
A
2J7L
1 NO
L
2J2Z
Nuc
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ome
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N d
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Pap
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Pap
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Pap
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Pap
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inhi
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wal
l or
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d bi
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esis
cell
wal
l or
gani
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n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
cell
wal
l or
gani
zatio
n an
d bi
ogen
esis
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wal
l or
gani
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n an
d bi
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X-r
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X-r
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NM
R
X-r
ay
X-r
ay
X-r
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X-r
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X-r
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X-r
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Sac
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1 PD
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1 Y6Z
1 IT
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1 FX
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3
2UW
J
2GZ
P
2FC
W
Pa
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apK
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X-r
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X-r
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NM
R
X-r
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X-r
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X-r
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X-r
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X-r
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X-r
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The
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1Q3R
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1Q3S
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AD
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1Q3Q
T
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AM
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The
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Ure
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10
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NM
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R
X-r
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M
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H
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