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Structure
Article
Design of a Phosphorylatable PDZ Domainwith Peptide-Specific Affinity ChangesColin A. Smith,1,8,* Catherine A. Shi,2 Matthew K. Chroust,3 Thomas E. Bliska,6 Mark J.S. Kelly,4 Matthew P. Jacobson,4,7
and Tanja Kortemme5,7,*1Graduate Program in Biological and Medical Informatics2Graduate Group in Biophysics3School of Dentistry4Department of Pharmaceutical Chemistry5Department of Bioengineering and Therapeutic Sciences
University of California San Francisco, 1700 4th Street, San Francisco, CA 94158, USA6Williams College, 880 Main Street, Williamstown, MA 01267, USA7California Institute for Quantitative Biosciences, 1700 4th Street, San Francisco, CA 94158, USA8Present address: Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry,
Am Fassberg 11, Gottingen, Germany*Correspondence: [email protected] (C.A.S.), [email protected] (T.K.)
http://dx.doi.org/10.1016/j.str.2012.10.007
SUMMARY
Phosphorylation is one of the most common post-translational modifications controlling cellular pro-tein activity. Here, we describe a combined com-putational and experimental strategy to design newphosphorylation sites into globular proteins to regu-late their functions. We target a peptide recognitionprotein, the Erbin PDZ domain, to be phosphorylatedby cAMP-dependent protein kinase. Comparing thefive successful designs to the unsuccessful cases,we find a trade-off between protein stability and theability to be modified by phosphorylation. In twodesigns, Erbin’s peptide binding function is modifiedby phosphorylation, where the presence of the phos-phate group destabilizes peptide binding. One ofthese showed an additional switch in specificityby introducing favorable interactions between adesigned arginine in the peptide and phosphoserineon the PDZ domain. Because of the diversity of PDZdomains, this opens avenues for the design of re-lated phosphoswitchable domains to create a reper-toire of regulatable interaction parts for syntheticbiology.
INTRODUCTION
Rational protein design has been increasingly successful at pro-
ducing proteins with new functions. Many early results involved
adapting an existing function for new uses, such as changing
enzyme substrate specificity (Wells et al., 1987; Yoshikuni
et al., 2006), modifying protein interaction specificity (Shifman
and Mayo, 2003; Kortemme et al., 2004; Grigoryan et al., 2009;
Kapp et al., 2012), or altering fluorescent properties (Treynor
et al., 2007). More recent efforts have shown remarkable prog-
ress in creating proteins with entirely new functions, including
54 Structure 21, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights
enzymatic activity (Jiang et al., 2008; Rothlisberger et al.,
2008), de novo protein binding (Karanicolas et al., 2011), bio-
mineralization catalysis (Masica et al., 2010), and oxygen trans-
port (Koder et al., 2009). Proteins have also been engineered to
adapt and respond to their environment in different ways, such
as pH-sensitive antibodies (Murtaugh et al., 2011), proteins
that change folds in response to pH or other ions (Cerasoli
et al., 2005; Palmer et al., 2006; Ambroggio and Kuhlman,
2006), small domains that fold or unfold upon phosphorylation
(Balakrishnan and Zondlo, 2006; Riemen and Waters, 2009),
phosphorylation-induced oligomerization of alpha helices (Szilak
et al., 1997; Signarvic and DeGrado, 2003), and FRET-based
reporters of protein kinase activity (Zhang et al., 2001). Such
designs can form the basis of synthetic, posttranslational regula-
tory machinery for programming cellular behavior.
Nature has evolved many mechanisms for modifying and
regulating protein function, with one of the most pervasive being
phosphorylation. One of the simplest regulatory mechanisms is
disruption of a protein-protein interface via addition of a phos-
phate group. Other proteins create interactions upon phosphor-
ylation, with domain families, such as SH2 and 14-3-3, having
specifically evolved for this purpose. Phosphorylation can also
alter intramolecular properties that change protein activity, with
phosphorylation of the kinase ‘‘activation loop’’ being a canonical
example (Steinberg et al., 1993; Adams et al., 1995).
Phosphorylation sites can be readily introduced into proteins
by fusion of an existing phosphorylatable domain or disordered
region. One of the first reported instances of a phosphorylation
site introduced through site-specific mutation was the incorpo-
ration of a cAMP-dependent protein kinase (PKA) recognition
sequence in the C-terminal tail of human interferon a (IFN-a) (Li
et al., 1989), with the goal of creating radioactive 32P-labeled
proteins for research and clinical use. That work was followed
by other examples, including mutation of the C-terminal region
of a monoclonal antibody to be recognized by casein kinase I
(CKI) (Lin et al., 1999). Another phosphosite was designed in
the semiflexible linker region between the Fab and Fc regions
of a monoclonal antibody heavy chain (Wu et al., 2004). Beyond
producing radiolabeled proteins, other phosphosite design
reserved
AKQEIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLLQPGDKIIQANGYSFINIEHGQAVSLLKTFQNTVELIIVREVSS
RRXSIKRXSI...
Scan Degenerate PKA Recognition Motif Over
91 PDZ Positions
Filter Destabilizing Mutations with
Rosetta
1235 Sequences91 Positions
148 Sequences29 Positions
71 Sequences23 Positions
8 Sequences8 Positions
Filter Sequences with pkaPS Phosphosite
Predictor
Filter by Visual Inspection
-18 PFRPDDDGIFVTRVQRRGSASKLLQPGDKIIQANGYSFINIE +23
Score 42 Residues Around Target Serine
Figure 1. Computational Phosphosite Design Protocol Applied and
Tested Here
Table 1. Scoring of Initial Phosphosite Designs
Protein
Mutant
Sequence
WT
Sequence
Rosetta D
Score
pkaPS
Score
S13 RKDSE EKDPE �5.50 0.96
S14 KRPSL KDPEL �1.54 1.31
S33 FRPSV FRPDD �0.44 1.09
S43 RRVSP TRVQP �0.12 1.76
S45 VRPSG VQPEG �3.17 1.08
S47 RRGSA PEGPA �0.72 2.06
S53 KRLSP KLLQP �0.50 1.37
S82 RRFSF KTFQN �0.58 1.15
Initial phosphosite designs based on having a negative change in Rosetta
score (relative to thewild-type) and a high pkaPS score.Mutated residues
are in bold.
Structure
Design of a Phosphoswitchable PDZ Domain
efforts have aimed at modulating protein-protein interactions
and protein regulation. Yeh and colleagues (Yeh et al., 2007)
mutated a peptide recognized by the syntrophin PDZ domain
so that it could also be phosphorylated by PKA. Upon phosphor-
ylation the PDZ-peptide interaction was destabilized, which
allowed activation of individual guanine exchange factors
(GEF) fused between the peptide and PDZ domain, whose inter-
action conformationally restricted and inactivated the GEF.
As illustrated above, most previous studies have focused on
introducing phosphorylation sites into sequences outside folded
protein domains or in triggering folding/unfolding transitions
upon phosphorylation (Balakrishnan and Zondlo, 2006; Riemen
and Waters, 2009). Here, we describe the work that involves
phosphorylation of a globular domain itself to modulate its func-
tion. This should enable new avenues for designing controllable
domain-domain interactions, enzyme activities, and conforma-
tional changes. In addition, characterization of design successes
and failures may provide clues about how phosphorylation
sites evolve in folded domains. We make use of the Erbin PDZ
domain, whose peptide interaction specificities have been ex-
tensively characterized with phage display (Laura et al., 2002;
Tonikian et al., 2008; Ernst et al., 2009). We first apply a compu-
tational design strategy to select mutations that are predicted to
not disrupt native binding activity in the unphosphorylated form
and to be phosphorylated by PKA. Guided by characteriza-
tion of thermostability, chemical shifts, and solvent exposure,
we then evaluate the successes and limitations of the design
strategy and generate several additional successful designs.
Finally, we characterize the changes in binding affinity and inter-
action specificity upon phosphorylation with a set of peptide
variants.
RESULTS
Initial Phosphosite DesignsWe first wanted to develop and test a computational strategy to
generate sites in the Erbin PDZ domain that could be phosphor-
ylated by cAMP dependent protein kinase (PKA), irrespective of
whether phosphorylation would affect peptide binding to the
PDZ domain. Exhaustive scanning of a degenerate PKA recogni-
tionmotif yielded over 1,000 potential sequences (see the Exper-
imental Procedures; Figure 1). We used the change in Rosetta
score (Smith and Kortemme, 2008) in the presence of the
peptide as a proxy for the extent of disruption in fold stability
or binding affinity. Our protocol incorporated limited amounts
of backbone flexibility shown to better predict side-chain con-
formations (Smith and Kortemme, 2008). To computationally
approximate the probability of phosphorylation, we used pkaPS
(Neuberger et al., 2007), a sequence-based predictor of phos-
phorylation sites optimized for PKA. Using those computational
tools and subsequent manual inspection of a reduced number of
candidates (Figure 1), we selected eight sequences for testing
(Figure 2; Table 1). Each designwas named for the serine residue
targeted for phosphorylation (e.g., S13 standing for serine 13).
Using a radiolabeled phosphotransfer assay, the eight designs
were tested for phosphorylation by PKA. S47 and S82 both
showed significant levels of phosphorylation, with a stronger
signal at S47 (Figure 3A). Using mass spectrometry, we con-
firmed that each domain was singly phosphorylated (i.e.,
Structure 21
M+80) and found that S47 was phosphorylated to completion
in the time used for the gel-based assays, whereas S82 was
not. Residue 47 is in the middle of a 15-residue loop between
the third and fourth beta strands of the Erbin PDZ domain (Fig-
ure 2B). Residue 82 is located on a short loop between the alpha
helix and the last beta strand. The pattern of phosphorylation did
not strictly follow the pkaPS scores: although S47 had the high-
est pkaPS score (2.1; Table 1), the designs with next three
highest scores (1.8–1.3) did not phosphorylate. However, S82,
the construct with the next lowest score (1.2), could be
phosphorylated.
Given that known phosphorylation sites are often found in
disordered regions of proteins (Iakoucheva et al., 2004), we
hypothesized that disorder and/or protein stability may play
a role in substrate phosphorylation. To determine whether this
was the case for our designs, we used apparent melting temper-
atures from thermal denaturation as a proxy for protein stability.
, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights reserved 55
S82S53S47S45S43S33S14S13WT
10 12 14 30 32 34 36 38 40 42 44 46 48 50 52 54 78 80 82 84
VVVVVVVVV
EEEEEEERE
KKKKKKKKK
DDDDDDRDD
PPPPPPPSP
EEEEEESEE
LLLLLLLLL
FFFFFFFFF
RRRRRRRRR
PPPPPPPPP
DDDDDSDDD
DDDDDVDDD
DDDDDDDDD
GGGGGGGGG
IIIIIIIII
FFFFFFFFF
VVVVVVVVV
TTTTRTTTT
RRRRRRRRR
VVVVVVVVV
QQQRSQQQQ
PPRPPPPPP
EERSEEEEE
GGGGGGGGG
PPSPPPPPP
AAAAAAAAA
SSSSSSSSS
KKKKKKKKK
LRLLLLLLL
LLLLLLLLL
QSQQQQQQQ
PPPPPPPPP
LLLLLLLLL
RKKKKKKKK
RTTTTTTTT
FFFFFFFFF
SQQQQQQQQ
FNNNNNNNN
TTTTTTTTT
... ... ... ...
A
B
Figure 2. Initial Phosphosite Designs
(A) Protein sequences of wild-type Erbin PDZ
domain and eight designed phosphorylation
candidates. Amino acids are numbered such that
residue 1 is the first amino acid present in the PDB
structure 1MFG (Birrane et al., 2003) (i.e., residue
1277 in full-length Erbin). Serines targeted for
phosphorylation are shown in red, added arginines
are shown in blue, and added hydrophobic amino
acids are shown in orange. See also Figure S1 for
gel filtration chromatography to characterize the
oligomeric state of WT and S47.
(B) Structural distribution of target serines shown
on PDB 1MFG (Birrane et al., 2003) with C-alpha
atoms shown as spheres and the bound peptide
shown in purple. All structure figures were
rendered with PyMOL (Schrodinger, LLC, 2012).
Structure
Design of a Phosphoswitchable PDZ Domain
Figure 3B shows melting curves measured by circular dichroism
for wild-type Erbin and the eight designs. S82 and S47, the two
variants that could be phosphorylated, also had the two lowest
melting temperatures of the initial eight designs (Table 2). These
lower melting temperatures relative to the wild-type protein were
unexpected (based on our design criterion to not destabilize the
protein) and indicate known difficulties in accurately estimating
the energetic effects of surface mutations. As discussed later,
these two designs were still functional and bound to all tested
peptides (Table 3). S47 showed a 4- to 6-fold reduction in
binding affinity over wild-type and S82 bound slightly tighter
than wild-type (less than a 2-fold increase). Reversible GuHCl
denaturation of WT and S47 mirrored the thermal denaturation
results and showed that S47 had lower fold stability than WT
(Figure S2 available online). The qualitative rates of phosphoryla-
tion do not necessarily correlate, as S82 was less thermostable
than the more rapidly phosphorylated S47.
Nuclear Magnetic Resonance CharacterizationTo investigate structural differences between wild-type and
S47, the mutant that showed the greatest rate of phos-
phorylation, we acquired HSQC spectra for it as well as
wild-type Erbin in the unbound form (Figure 4A). Although
assignments were previously determined for wild-type Erbin
bound to peptide TGWETWV (Skelton et al., 2003), there
56 Structure 21, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights reserved
were significant differences between it
and the free form, preventing assign-
ment transfer. Therefore, we obtained
de novo 1H, 15N, 13CA, and 13CB reso-
nance assignments for both WT and
S47 in the unbound form. In Figure 4A,
peaks for equivalent residues in WT
and S47 are connected by lines. Also,
as indicated, the spectrum for S47 had
two additional 1H-15N peaks because
of the P44R and P47S mutations. Fig-
ure 4B shows atom-specific chemical
shift differences, as well as the overall
chemical shift difference normalized by
the standard deviation (s) of all reso-
nances found in a database of protein chemical shifts (Ulrich
et al., 2008). As expected, the most extensive chemical shift
changes (up to 1s) were for residues 42–52, which are next
to the mutated residues in sequence. Interestingly, a segment
of residues adjacent in the tertiary structure, 7–17, also had
large shift differences of up to 0.7s (Figure 4C), and a more
distant segment, 82–85, had smaller differences up to 0.3s.
These differences suggest that there was a change in structure
and/or dynamics that was more than would be expected for a
mutation of several solvent-exposed residues.
To accommodate recognition by PKA, there is likely some
degree of local or global structural opening/unfolding that is
either a transient or stable feature of the solution ensemble.
We reasoned that such opening is likely associated with an in-
creased solvent exchange of amide hydrogens. Using CLEANEX
hydrogen exchange experiments (Hwang et al., 1998) with
mixing times from 10–100 ms, we observed residues in all
three segments mentioned above having greater hydrogen
exchange in S47 than in WT (Figure 5). These changes in solvent
exchange are among the most significant observed over the
whole protein structure. Assuming the hydrogen exchange
profiles of the other nonphosphorylatable designs are similar to
WT, this supports a model in which S47 is shifted toward
a more open conformation at equilibrium, possibly contributing
to recognition and phosphorylation by PKA.
30 40 50 60 70 80
Nor
mal
ized
Elli
ptic
ity1.
00.
50.
0 S82S47S33S53S43S45S13S14WT
WT S13 S14 S33 S43 S45 S47 S53 S82A
B
Figure 3. Phosphorylation and Thermostability of Initial Designs
(A) SDS-PAGE gel (10%–20%) of Erbin PDZ domain designs phosphorylated
with 32P ATP.
(B) Circular dichroism signal at 218 nm showing thermal denaturation of
designs. Normalization is described in the Experimental Procedures. See also
Figure S2 for equilibrium GuHCl denaturation curves of WT and S47.
Table 2. ApparentMelting Temperatures of Phosphosite Designs
Initial Designs Secondary Designs
Protein Tm,app Protein Tm,app
S82 44 S13-A47 43
S47 46 S43-A47 44
S33 49 S14-A47 44
S53 50 S45-A47 48
S43 57 S47A 50
S45 58 S13-A47a 50
S13 60 S14-A47a 51
S14 61 pS13-A47a 55
WT 66 pS14-A47a 55
Temperatures (Tm,app) in degrees Celcius from irreversible thermal dena-
turation measured by circular dichroism signal at 218 nm.a6xHis tag removed. Phosphorylated designs are indicated by ‘‘p’’.
Table 3. Binding Affinities of Unphosphorylated and
Phosphorylated Designs
Peptides
Protein TGWETWV TGWETRV TGWETDV
S13-A47 0.022 ± 0.0025 1.4 ± 0.03 0.71 ± 0.11
pS13-A47 0.072 ± 0.0016 4.5 ± 0.61 3.2 ± 0.65
S14-A47 0.017 ± 0.0011 3.3 ± 0.07 0.54 ± 0.10
pS14-A47 0.17 ± 0.032 22 ± 2.3 4.7 ± 1.3
S43-A47 0.050 ± 0.0023 26 ± 0.3 16 ± 0.2
pS43-A47 0.45 ± 0.034 37 ± 2.8 123 ± 7.7
S47 0.094 ± 0.018 28 ± 0.6 21 ± 0.6
pS47 0.14 ± 0.003 25 ± 1.1 25 ± 0.8
S82 0.017 ± 0.0008 3.2 ± 0.03 1.9 ± 0.12
pS82 0.024 ± 0.0028 4.5 ± 0.22 3.1 ± 0.04
WT 0.022 ± 0.0016 5.2 ± 0.30 3.5 ± 0.18
Dissociation constants (Kd) in mM derived from fluorescence polarization
experiments for unphosphorylated and phosphorylated PDZ domains
with three peptides differing at the �1 position. Standard errors from
multiple independent experiments are given. See also Figure S5 for indi-
vidual binding curves. Phosphorylated designs are indicated by ‘‘p’’.
Structure
Design of a Phosphoswitchable PDZ Domain
Second Round of Phosphosite DesignsAfter noting the chemical shift differences in segments 7–17 and
42–52 of S47, we hypothesized that the structural changes
enabling S47 to be phosphorylated might also rescue phosphor-
ylation at other sites located in the altered regions. Furthermore,
the increase in S47 hydrogen exchange at residues 15, 45, and
46 suggested that the changes might be related to structural
opening. Therefore, we designed a different template protein,
A47, which included the arginine mutations of S47 (P44R and
E45R) but incorporated an alanine at position 47 to prevent
phosphorylation. Using that template, we reintroduced the
mutations for the previous S13, S14, S43, and S45 designs (Fig-
ure 6A). These candidate phosphosites were chosen because
they were located on the structurally perturbed loops and in
regions near the peptide that were more likely to affect binding
affinity.
Both radiolabeled phosphotransfer assays and mass spec-
trometry indicated that A47 did not phosphorylate (Figure 6B),
as expected, showing that phosphorylation of S47 was specific
to serine 47 and that the A47 construct was suitable for addition
of other sites. The same assays showed that three out of the four
constructs (S13, S14, and S43) could now be phosphorylated in
the A47 background (Figure 6B). Determination of the thermo-
stability using circular dichroism showed that A47 was more
stable than S47 but still significantly less stable than WT. All of
the secondary phosphosite designs were less stable than the
A47 construct on which they were based, and the three phos-
phorylatable designs were less stable than S47.
Beyond stability (Table 2 lists apparent melting temperatures)
other sequence-based factors could play a role in the failure of
the other designs. S33, S45–A47, and S53 had apparent melting
temperatures in the 48�C–50�C range that were close to the
other phosphorylatable designs. However, although all have an
arginine residue two sequence positions before the serine,
none of them have an arginine three positions before, likely
Structure 21
reducing their recognition by PKA, which strongly prefers two
upstream arginines at the �3 and �2 positions (Kemp et al.,
1977; Hutti et al., 2004).
In the context of the present study, it is difficult to fully decon-
volute all of the sequence and structure-based aspects of phos-
phorylation, in particular the relative importance of local versus
global instability. Although thermal denaturation was performed
on all designs, more extensive nuclear magnetic resonance
(NMR) characterization of solvent exposure was limited to WT
and S47. Nevertheless, the locally destabilizing effects of the
S47 mutations, along with their ability to rescue the phosphory-
lation of S13, S14, and S43, suggests that local substrate desta-
bilization may play an important role in phosphorylation.
Binding Affinity Changes upon PhosphorylationTo determine how the engineered mutations and phosphoryla-
tion affected PDZ domain function, we measured dissociation
, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights reserved 57
A
B
C Figure 4. Chemical Shift Differences
between WT and S47
(A) 2D 15N HSQC of wild-type (blue) and S47 (red)
Erbin PDZ domains with equivalent residues con-
nected by black lines and residues mutated from
proline given numbered labels. See also Figure S3
for spectra at multiple concentrations.
(B) Normalized overall (see the Experimental
Procedures) and atom-specific chemical shift
differences between wild-type and S47.
(C) Overall chemical shift differences displayed on
the PDB structure 1MFG (Birrane et al., 2003) with
C-alpha radii scaled by 50%–100% to reflect nor-
malized overall chemical shift difference. Mutated
residues, not contained in the plots, are shown in
purple. The peptide (not included in the NMR ex-
periment) is shown in purple for referencepurposes.
Structure
Design of a Phosphoswitchable PDZ Domain
constants (Kd) for three synthetic peptides (Figure 7A). The first
peptide was a phage-display derived (Laura et al., 2002) high-
affinity sequence (TGWETWV), whose residue positions are
given decreasing numbers starting at zero for the C-terminal
valine. Because the side chain of the�1 position had the closest
proximity to the phosphosite positions (Figure 7B), we hypothe-
sized that electrostatic interactions with the phosphate group
may be possible. Therefore, we also tested peptides with an
arginine (TGWETRV) and aspartate (TGWETDV) at the �1 posi-
tion to evaluate potential electrostatic attraction and repulsion.
Wild-type Erbin had the highest affinity for TGWETWV (22 nM)
and bound approximately two orders of magnitude more weakly
to peptides with the arginine and aspartate residues at the �1
position (see Table 3 and all binding curves in Figure S5).
Depending on the position, mutations in the designed Erbin vari-
ants either increased or decreased the base (unphosphorylated)
58 Structure 21, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights reserved
binding affinity to peptides. S43–A47 and
S47 bound the three peptides somewhat
weaker than wild-type Erbin by a factor
of up to six, whereas S13–A47, S14–
A47, and S82 all showed improvements
in binding affinity to the TGWETRV and
TGWETDV peptides by up to 6.5-fold.
The mutations in S13–A47, S14–A47,
and S82 were spatially clustered around
the peptide’s negatively charged C
terminus. Each involved the addition of
1–2 arginine residues, which could result
in electrostatic attraction and increased
binding. Subtle changes in the binding
site geometry could also account for the
observed affinity changes.
Upon phosphorylation, the S14–A47
and S43–A47 designs showed nearly
10-fold decreases in binding affinity (Fig-
ure 7A), corresponding to a change in
binding energy of 1.4 kcal/mol. With a
3- to 4-fold decrease, S13–A47 was less
perturbed by phosphorylation. S47 and
S82 both showed negligible decreases
in binding affinity. For S13–A47 and
S14–A47, which showed significant decreases in binding affinity
for all peptides, phosphorylation increased thermostability
(Figure 6D), suggesting that fold stability was not a factor in de-
stabilizing its binding interface. Instead, the degree of binding
affinity change appears to be associated with distance of
the phosphate group from the peptide with residues 14 and 43
being closer and residues 47 and 82being further (see Figure 7B).
The observed changes in binding affinity were similar for both
the tryptophan and aspartate peptides, indicating that there
was little additional electrostatic repulsion resulting from the
aspartate peptide. Double mutant cycle analysis in designed
beta-hairpin peptides has shown that interactions between adja-
cent phosphoserine/tryptophan residues were destabilizing,
whereas phosphoserine/valine were not (Riemen and Waters,
2009). The peptide binding affinities for S43–A47 show a similar
pattern of phosphoserine/tryptophan destabilization, along with
A C
B
Figure 5. Increased Amide Hydrogen Exchange around Phosphorylation Site
(A) Fitted CLEANEX (Hwang et al., 1998) hydrogen exchange curves (solid lines) showing increased solvent accessibility in the S47 design (red) over wild-type
(blue) for residues near the phosphorylation site. The ratio of peak volume (V) to reference peak volume (V0) is shown. Initial velocities are shownwith dashed lines
along with their standard errors (shaded regions).
(B) Difference in initial velocities (k) normalized by the sum of their standard errors (SEk). Points outside of the dashed region have nonoverlapping error bars.
Residues shown in (A) are boxed.
(C) Normalized initial velocity differences displayed on the PDB structure 1MFG (Birrane et al., 2003) with amide hydrogen radii scaled by 50%–100% to reflect
normalized differences in initial velocities. The peptide (not included in the experiment) is shown in purple for reference purposes.
Structure
Design of a Phosphoswitchable PDZ Domain
equivalent interface destabilization from the phosphoserine/
aspartate interaction. However, for S43–A47, the arginine
peptide showed only a marginal decrease in binding affinity
upon phosphorylation. This may indicate that electrostatic or
hydrogen-bonding interactions between the phosphate and
arginine (Figure 7C) largely counteract any destabilizing effects
of phosphorylation. Hydrogen bonds between phosphorylated
amino acids and arginine have been theorized to be particularly
strong (Mandell et al., 2007). For the arginine and aspartate
peptides, this differential destabilization results in a change in
specificity from preferring aspartate in the unphosphorylated
form (R: 26 mM versus D: 16 mM) to arginine in the phosphory-
lated form (R: 37 mM versus D: 123 mM).
DISCUSSION
This study highlights the successful incorporation of phosphory-
lation sites into globular protein structures. The most efficiently
phosphorylated initial design (S47) had decreased thermosta-
bility and increased backbone solvent exposure. By transferring
destabilizing mutations from that design to others, phosphoryla-
tion activity was rescued in three other designed proteins. Upon
phosphorylation, several designs showed approximately 10-fold
reductions in binding affinity and one design exhibited a change
in peptide binding specificity.
Structure 21
The interactions between natural PDZ domains and their
target peptides are sometimes controlled by phosphorylation,
with the most prevalent mechanism being phosphorylation of
the recognized peptide (Ivarsson, 2012). In addition to cognate
peptide phosphorylation, phosphorylation of PDZ domains
themselves occurs naturally and is known to disrupt peptide
binding. One site was found in the first PDZ domain of human
disks large homolog 1 (DLG1-1), which is phosphorylated at
S232 (Gardoni et al., 2003) (aligning with Erbin PDZ P13 or
E14) by Ca2+/calmodulin-dependent protein kinase II (CaMKII).
Another disruptive phosphorylation site is located on the first
PDZ domain of Npt2a-binding protein sodium-hydrogen ex-
changer regulatory factor-1 (NHERF1-1) that is phosphorylated
at S77 (Weinman et al., 2007) (aligning with Erbin PDZ S76).
The Erbin PDZ domain used in this phosphosite design study
is not known to be naturally phosphorylated.
Phosphorylation has been traditionally thought of as a mecha-
nism to modulate a single function, either through binary on/off
switching or a graded response (Pufall et al., 2005). The phos-
phoswitchable PDZ domains described here suggest that it is
possible to design proteins that switch from one function to
another upon phosphorylation. Although there are several
examples of peptide phosphorylation resulting in changes in
binding from one peptide recognition domain to another (Akiva
et al., 2012), changes in specificity of the peptide recognition
, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights reserved 59
30 40 50 60 70 80
Nor
mal
ized
Elli
ptic
ity1.
00.
50.
0 S13 A47S43 A47S14 A47S82S47S45 A47A47WT
A47S13A47
S14A47
S43A47
S45A47 S47 S82
S45 A47S43 A47S14 A47S13 A47
A4710 12 14 38 40 42 44 46 48
VVVVV
EEERE
KKKKK
DDRDD
PPPSP
EESEE
LLLLL
FFFFF
VVVVV
TRTTT
RRRRR
VVVVV
RSQQQ
RRRRR
SRRRR
GGGGG
AAAAA
AAAAA
... ... ...
B
C
A
30 40 50 60 70 80
Nor
mal
ized
Elli
ptic
ity1.
00.
50.
0 S13 A47 ( 6xHis)S14 A47 ( 6xHis)pS13 A47 ( 6xHis)pS14 A47 ( 6xHis)
D
Figure 6. Phosphorylation and Thermostability of Secondary
Designs
(A) Phosphorylation candidate designs based on combining a destabilized
alanine mutant of the S47 variant (A47) with previous designs (S13, S14, S43,
and S45; see Figure 2) containing the desired phosphorylation site near the
peptide-binding site.
(B) SDS-PAGE gel (10%–20%) showing phosphorylation of three of the
second round designs with 32P ATP.
(C) Circular dichroism signal at 218 nm during thermal denaturation shows that
phosphorylatable designs unfold at the same temperature or lower than S47.
(D) Thermal denaturation shows that phosphorylation stabilizes both S13-A47
and S14-A47. See also Figure S4 for thermal denaturation of His-tagged
S14-A47 incubated with TCEP.
Fol
d C
hang
e in
Kd
upon
Pho
spho
ryla
tion
02
46
810
S13 A47 S43 A47 S82S14 A47 S47
1 TGWETWVTGWETRVTGWETDV
A
B
C
R-1
pS43
Figure 7. Peptide-Dependent Changes in Binding Affinity upon
Phosphorylation
(A) Peptide binding affinity decreases upon phosphorylation shown as fold
changes in Kd upon phosphorylation (i.e., Kd,phosphorylated/Kd,unphosphorylated).
Three variants of a phage display derived high affinity peptide (Laura et al.,
2002) were tested, each with a different amino acid (W, R, or D) at the �1
position. Error bars reflect propagated standard errors from Table 3.
(B) C-alpha atoms of the phosphorylated residues and the�1 peptide position
are shown with spheres on the PDB structure 1MFG (Birrane et al., 2003).
(C) Computational model of phosphorylated S43-A47 bound to the TGWETRV
peptide built bymutating and optimizing side chains of the PDB structure 1N7T
(Skelton et al., 2003).
Structure
Design of a Phosphoswitchable PDZ Domain
domains themselves has not been readily observed. The in vitro
change in specificity we demonstrate is relatively modest, and
increases in affinity modulation may be required to produce
observable in vivo effects. With further design iterations, it
may be possible to increase the degree of change in specificity
shown here.
We observed an apparent inverse relationship between
protein stability and phosphorylation. Considerable evidence
already exists that enzymes trade off fold stability in order to opti-
mize their active sites for function (Shoichet et al., 1995) and that
60 Structure 21, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights
evolution of new functions requires either an excess buffer of
stability or compensatory stabilizing mutations (Tokuriki et al.,
2008). This study illustrates a similar stability-function trade-off
reserved
Structure
Design of a Phosphoswitchable PDZ Domain
in enzyme substrates, where instability appears to be correlated
with function, that is, the ability to be phosphorylated. Moreover,
it has been theorized that evolutionary pressure exists against
protein overstabilization because it makes proteins difficult to
regulate through the cell’s natural degradation machinery
(DePristo et al., 2005). In the case of our designed PDZ domains,
overstabilization would prevent regulation through posttransla-
tional modification. It stands to reason that natural proteins
may have similar constraints to facilitate chemical modifications
required for regulatory or other functional purposes.
For several pairs of designs in this study (i.e., S13/S13-A47
and S14/S14-A47), the sequence differences that enabled phos-
phorylation were 30 residues away from the phosphorylation
site in primary sequence. Predictors of phosphorylation that
only incorporate knowledge about local sequence context and
domain boundaries are incapable of discriminating the rate of
phosphorylation in these pairs. Instead, the most obvious
discriminating factor appears to be protein stability. Without
invoking protein stability, an alternative explanation could be
that surface and/or net-charge effects resulting from two addi-
tional arginine residues near the phosphorylation site promote
recognition by PKA.
It is likely that future studies will benefit from making small
decreases in protein stability a goal of the design process. For
this study, the computational screening process aimed to avoid
destabilization of the globular protein structure. Neither the
computational predictions nor visual inspection of the primarily
solvent-exposed mutations suggested that the PDZ domains
would be significantly destabilized. Although it has been shown
that optimization of charge-charge interactions can stabilize
proteins (Strickler et al., 2006), the stabilizing or destabilizing
effect of adding surface charges is still not well understood.
An advantageous aspect of the protein design strategy used
here is that there is relatively little overlap between the rede-
signed residues and those residues thought to bemost important
for determining PDZ-peptide specificity (Tonikian et al., 2008).
Because of this, the mutations used to introduce a PKA phos-
phorylation site into the Erbin PDZ domain could likely be trans-
ferred to other PDZ domains without significantly perturbing their
specificities. Because of the large natural diversity of PDZ
domain specificities (Tonikian et al., 2008), and the ease with
which other specificities can be synthetically created (Ernst
et al., 2009), it may be possible to expand this work into a reper-
toire of regulatable protein interaction parts for application in
synthetic biology. Such PDZ domains could be incorporated
into protein scaffolds to posttranslationally control or divert
cellular signal transduction (Good et al., 2011) or metabolic flux
through biosynthetic pathways (Dueber et al., 2009).
EXPERIMENTAL PROCEDURES
Phosphosite Design
For the initial round of phosphosite selection, we targeted the entire Erbin PDZ
domain protein sequence (Figure 1). The cAMP-dependent protein kinase
(PKA) recognition motif was represented as a five-residue sequence with
degeneracies in the first and last positions, R/K/X-R-X-S-I/K/F/V/X, with X
being the wild-type amino acid. The 15 possible combinations of this motif
were scanned over the 95-residue sequence taken from the Protein Data
Bank (PDB) code 1MFG (Birrane et al., 2003). The 91 possible positions yielded
1,235 unique candidate sequences. To limit destabilization of both the protein
Structure 21
fold and peptide binding, we only selected sequences whose total Rosetta
score was less than wild-type (i.e., predicted to be no less stable), which
was determined by applying the Backrub point mutation protocol (Smith and
Kortemme, 2008) to PDB code 1MFG with the peptide included. This resulted
in 148 sequences (at 29 positions). To take the context of the surrounding
protein sequence into account, these sequences were then screened for the
likelihood of phosphorylation with the pkaPS (Neuberger et al., 2007)
sequence-based PKA substrate predictor. This left 71 sequences (at 23 posi-
tions) with a pksPS scores greater than one. The candidate sequence with the
best pkaPS score at each position was visually inspected, and of these, the
most promising eight sequences (balancing kinase accessibility and closeness
to the canonical PKA recognition motif) that required six or fewer base pair
changes were selected for experimental screening. Kinase accessibility was
judged primarily by side-chain solvent exposure and how convex (as opposed
to concave) the structure was around the motif residues. This was balanced
against the number of arginine residues at the �2 and �3 positions (i.e.,
more convex sites were allowed to have fewer arginines).
These eight designs were named for the serine position that was targeted for
phosphorylation (i.e., S13 for serine 13). In S13 and S14, lysine 11 was origi-
nally designed to be mutated to arginine. Because mutation of this conserved
lysine has been shown to disrupt both PDZ domain stability and peptide
binding affinity (Harris et al., 2003), it was reverted to wild-type in both designs.
This resulted in a pkaPS score slightly lower than the one for S13, indicating
a slightly reduced predicted probability of phosphorylation. The reduction in
the number of arginine residues and the largely buried lysine were allowed
because of the convex, almost wedge-like shape of the PDZ domain around
these two positions.
Cloning and Mutagenesis
The Erbin PDZ domain (base pairs 3943–4239 of the coding sequence) was
cloned out of full-length Homo sapiens Erbin (GenBank 358679310:311-
4549) that had been previously cloned into pRK5-myc (Borg et al., 2000)
(courtesy of J.P. Borg). This Erbin construct was inserted along with an
N-terminal ATG between the NotI and XhoI sites of a modified pET-47b(+)
expression vector (Novagen, Darmstadt, Germany) that had eight base pairs
(GGGTACCA) after the HRV-3C cleavage site removed (courtesy R.M. Stroud).
For NMR studies, a shortened construct was prepared by inserting it without
the additional N-terminal ATG between the EcoRI and XhoI sites of the modi-
fied pET-47b(+) expression vector.Mutations weremadewith theQuikChange
mutagenesis kit (Agilent, Santa Clara, CA, USA) and primers designed
with the online QuikChange primer design tool (http://www.agilent.com/
genomics/qcpd).
Protein Expression and Purification
All proteinswere expressed in BL21 cells at 25�C, inducedwith 0.5mM IPTG at
OD 0.5 (600 nm), and harvested after 6 hr. Following a standard protocol
(QIAGEN, 2003), cells were lysed after storage at �80�C in 300 mM NaCl,
10 mM imidazole, and 50 mM Na2HPO4 (pH 8) and then sonicated and spun
down. After incubation of the supernatant for 1 hr with Ni-NTA agarose resin
(QIAGEN, Hilden, Germany), beads were spun down and washed three times
with 300 mM NaCl, 20 mM imidazole, and 100 mM Na2HPO4 (pH 8), followed
by elution with 300 mM NaCl, 250 mM imidazole, and 50 mM Na2HPO4 (pH 8).
Protein concentrations were determined with the Pierce Coomassie Plus
Bradford Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA) using
the included bovine serum albumin (BSA) as the standard.
Erbin constructs were expressed in Luria broth (LB) medium, purified, dia-
lyzed into 300 mM NaCl and 50 mM Na2HPO4 (pH 8), and then stored at
4�C. Mass spectrometry analysis of S43-A47 suggested that the five rare
AGA/AGC arginine codons were resulting in misincorporation of one lysine
(mass loss of 28 daltons, M-28; Calderone et al., 1996) in 30%–40% of the
expressed protein. Cotransformation of pET-47 S43-A47 with the pJY2
plasmid (You et al., 1999) (Enzo Life Sciences, Farmingdale, NY, USA) amelio-
rated the effect. For NMR studies, Erbin constructs were expressed in
M9 minimal medium (6 g/l Na2HPO4, 3 g/l KH2PO4, 0.5 g/l NaCl, 247 mg/l
MgSO4, and 14.7 mg/l CaCl2) with 1 g/l 15N NH4Cl (Isotec, St. Louis, MO,
USA) and 4 g/l glucose. Doubly labeled proteins were expressed by
substituting 1 g/l 13C glucose (Isotec). After purification, labeled proteins
were dialyzed into PBS at a pH of 6.5, similar to a previous NMR study (Skelton
, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights reserved 61
Structure
Design of a Phosphoswitchable PDZ Domain
et al., 2003), and stored at 4�C. The constructs used for NMR studies were
shown to be monomeric up to 750 mM by high-performance liquid chromatog-
raphy (HPLC) gel filtration (Figure S1) and comparison of one-dimensional
(1D)/two-dimensional (2D)NMRspectra at different concentrations (FigureS3).
The Mus musculus PKA catalytic subunit was expressed using a previously
constructed pET-15b vector (Narayana et al., 1997) (Addgene 14921). Purified
PKA was dialyzed into 200 mM KCl, 5 mM DTT, and 20 mM KH2PO4 (pH 6.5)
and then stored at 4�C.
Protein Constructs
After expression, the longer construct cloned into the NotI and XhoI sites
was 137 amino acids in length, with wild-type having the sequence of
AHHHHHHSAALEVLFQGPGSEFCTGLGAPDVRRQACGRMHELjAKQEIRVRV
EKDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLLQPGDKIIQANG
YSFINIEHGQAVSLLKTFQNTVELIIVREVSS (the vertical bar immediately
precedes the alanine assigned residue number 1). It was used for radiolabeled
phosphoassays and circular dichroism studies. For binding studies, the 6xHis
tag of this construct was cleaved (see below), giving the 121 amino acid
wild-type sequence GPGSEFCTGLGAPDVRRQACGRMHELjAKQEIRVRVE
KDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLLQPGDKIIQANG
YSFINIEHGQAVSLLKTFQNTVELIIVREVSS. For NMR studies, the shorter
construct cloned between EcoRI and XhoI yielded a 120 amino acid wild-
type sequence, AHHHHHHSAALEVLFQGPGSEFHELjAKQEIRVRVEKDPEL
GFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLLQPGDKIIQANGYSFINI
EHGQAVSLLKTFQNTVELIIVREVSS.
Phosphorylation Assays
Phosphorylation assays were conducted with 80 mM g-32P ATP (0.012 mCi/ml),
40 mMPDZ domain, 0.4 mMPKA, 2mMDTT, 10mMMgCl2, 300mMNaCl, and
50 mM Na2HPO4 (pH 8). After incubation at 33�C for 1 hr, the reactions were
quenched by addition of Laemmli sample buffer and boiling at 95�C. 10%–
20% Tris-HCl SDS-PAGE gels (BioRad, Hercules, CA, USA) were imaged
using a storage phosphor screen. The number of covalently attached phos-
phates for all proteins was determined via mass spectrometry using a Waters
Micromass LCT Premier LC-MS system.
Circular Dichroism Spectroscopy
Thermal denaturation was performed on a Jasco J-715 spectrometer using
a 1 mm cuvette. Erbin constructs were diluted to 40 mM with 300 mM NaCl
and 50 mM Na2HPO4 (pH 8). Data acquisition was done at 218 nm (the peak
of the native spectrum) using a bandwidth of 1 nm, response time of 16 s,
temperature slope of 1�C/minute, and data pitch of 0.5�C. Between samples,
cuvettes were washed and stored in aqua regia (1:3 solution of concentrated
nitric acid and hydrochloric acid). In other experiments, thermal denaturation
of wild-type Erbin was determined to be irreversible.
Melting data having temperature (t) and ellipticity signal (y) were fit in R
(R Development Core Team, 2012) with the equation
y =yhigh � ylow
1+ eðTm;app�tÞ=s + ylow + at:
The fitted parameters included apparent melting temperature (Tm,app), low
and high values of the signal (ylow and yhigh, respectively), scale of the unfolding
transition (s), and linear background slope (a). Because all curves displayed
similar linear slopes before and after the unfolding transition, the a slope
parameter was fit globally across all protein curves. For visual clarity, plots
were normalized such that ylow = �1, yhigh = 0, and a = 0.
NMR Spectroscopy1H-15N HSQC spectra for WT and S47 were acquired on a Bruker 800 MHz
spectrometer and processed with Bruker Topspin. For all experiments, the
target sample temperature was set to 300.7 K at the console. Complete reso-
nance assignments for both WT (Biological Magnetic Resonance Data Bank
[BMRB] entry 18785) and S47 (BMRB entry 18786) were done with a combina-
tion of HNCA, HNCACB, and CBCA(CO)NH experiments on a 500 MHz Bruker
spectrometer. Spectra were processed in NMRPipe, and assignments were
carried out in CcpNmr Analysis. To determine normalized overall chemical shift
differences between WT and S47, the differences were first divided by the
residue type and atom-specific standard deviations from the Biological
62 Structure 21, 54–64, January 8, 2013 ª2013 Elsevier Ltd All rights
Magnetic Resonance Data Bank (BMRB) statistics stored in CcpNmr Analysis.
The overall difference was then calculated by taking the root-mean-square of
all normalized differences for a given residue.
CLEANEX hydrogen exchange spectra (Hwang et al., 1998) were acquired
on a Bruker 800 MHz spectrometer in separate experiments with mixing times
of 10, 20, 40, 60, 80, and 100 ms. To determine reference peak intensities and
ensure sample homogeneity, a standard Fast-HSQC (FHSQC) was acquired
immediately before and after the series of CLEANEX experiments. WT Erbin
showed negligible precipitation, so all mixing times were acquired in a single
day using a 900 mM sample with eight scans per phase cycle. To compensate
for an increased rate of S47 precipitation, especially at high concentrations,
the different mixing times were acquired over two days (10/40/80 and 20/60/
100 ms, respectively) using identical 400 mM samples with 16 scans per phase
cycle. Similar data was obtained with a 900 mM S47 sample and eight scans
per phase cycle, albeit with considerably more noise.
To control for sample precipitation, a 1D proton spectrum was acquired
before and after each 2D spectrum. Normalization factors for all 1D spectra
were determined by calculating the scaling factor that gave the best least-
squares superimposition of the first 1D spectrum onto all subsequent spectra
over the 9.6–6.6 and 2.6–0.1 ppm ranges. Normalization factors for each 2D
spectrum were determined by averaging the normalization factors of the 1D
spectra immediately before and after. S47 showed a decrease in intensity of
approximately 50% over the course of each day.
Spectra were processed with NMRPipe, and peak volumes were integrated
with CcpNmr Analysis. CLEANEX hydrogen exchange buildup data consisting
ofmixing times (tm), peak volumes (V), and reference peak volumes (V0) were fit
in R (R Development Core Team, 2012) with the equation (Hwang et al., 1998)
V
Vo
=k
R1A;app + k � R1B;app
�e�R1B;apptm � e�ðR1A;app + kÞtm �:
With very low exchange rates (k), the apparent longitudinal/transverse relax-
ation rate (R1A,app) does not significantly affect the shape of the buildup, which
leads to unstable curve fitting. For those amide protons, R1A,app was fixed at
0 s�1. The previously determined value (Hwang et al., 1998) of 0.6 s�1 was
used for the apparent relaxation rate of water (R1B,app).
Preparation of Phosphorylated Protein
Phosphorylated protein was prepared by addition of 5 mM ATP, 1 mM TCEP,
and 10 mM MgCl2. Phosphorylation during the reactions was monitored by
mass spectrometry. Depending on the phosphorylation rate, PKA was added
up to10%of the total proteinmassand incubated at 4�C–16�C.Uponcomplete
phosphorylation, several of the proteins showed two phosphorylation events
(M+160). Analysis of the protein sequences with pkaPS suggested the second
most probable PKA phosphorylation site to be a serine immediately after the
N-terminal 6xHis tag. Cleavage of the tag with HRV-3C protease (courtesy
R.M. Stroud) confirmed this hypothesis, so the 6xHis tags of all unphosphory-
lated and phosphorylated PDZ domains were cleaved prior to binding studies.
The 6xHis tag, His-tagged PKA, and HRV-3C protease were removed using
TALON His-Tag purification resin (Clontech, Madison, WI, USA).
Fluorescence Polarization
Three peptides, TGWETXV, each with a different amino acid at the X position
(W, R, or D) were synthesized with an N-terminal fluorescein label (GenMed
Synthesis, San Antonio, TX, USA; quality control using mass spectrometry
and purity >85% via C18 reverse-phase HPLC). Lyophilized peptide was
resuspended in 5 mM Na2HPO4 (pH 7.4) and the peptide concentration
was determined using the fluorescein extinction coefficient at 490 nm
(76,000 M�1 cm�1) (Mota et al., 1991).
Fluorescence polarization assays were performed by preparing solutions
with log-spaced concentrations of PDZ domain over two orders magnitude.
The assay solutions were then made up with 40% (by volume) of the PDZ dilu-
tions, 0.25%1 mMpeptide, 49.75%binding buffer (20mMDTT, 20mMHEPES
[pH 7.4]) (Lauffer et al., 2010), and 10% 1mg/ml BSA. This resulted in a 2.5 nM
final peptide concentration and 120 mM NaCl. Solutions were prepared on
96-well plates, and three aliquots per dilution were then transferred to
384-well plates for reading on an Analyst AD fluorometer (Molecular Devices,
Sunnyvale, CA, USA). To prevent protein precipitation, mixing was done in
a cold room, and plates kept on ice until immediately prior to reading after
reserved
Structure
Design of a Phosphoswitchable PDZ Domain
equilibration to 25�C. All measurements were done in at least duplicate using
separate batches of expressed protein and separate phosphorylation reac-
tions. Binding measurements for pairs of unphosphorylated and phosphory-
lated proteins taken from same expression batch were done on the same
day, and replicates of pairs were done on different days.
Fluorescence polarization data were first normalized by subtracting the
mean polarization for a blank sample with no PDZ domain. The resulting
changes in polarization (Y) and concentrations (D) were fit in R (R Development
Core Team, 2012) with the following equation
Y =Y0
D
D+Kd
:
The fit parameters included the dissociation constant (Kd) and the maximum
change in polarization (Y0). For a given Kd (A) with standard deviation (sA) and
N replicates, the standard error (SEA) was determined, SEA = sA=ffiffiffiffiN
p. For
a given fold change between two Kd values (A/B) and correlation coefficient
between A and B (rAB), the standard error was calculated using typical prop-
agation of uncertainty,
SEA=B =A
B
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðsA=AÞ2 + ðsB=BÞ2�2rABsAsB=ðABÞ
qffiffiffiffiN
p :
Phosphoserine Modeling
The interaction between the phosphoserine of S43-A47 and the TGWETRV
peptide was modeled using PDB code 1N7T (Skelton et al., 2003), an NMR
structure of wild-type Erbin bound to peptide TGWETWV. PDZ residues 40,
43, 44, 45, and 47, as well as the peptide residue �1 were first changed
to the phosphorylated target sequence of S43-A47 and TGWETRV with
PyRosetta (Chaudhury et al., 2010). Those side chains, along with adjacent
residue R41, were then optimized with the Protein Local Optimization Program
(PLOP) (Groban et al., 2006) version 17.0, using the ‘‘side predict’’ command
with minimum overlap factor of 0.5.
ACCESSION NUMBERS
The BMRB entries for the chemical shift data reported in this paper are 18785
(Erbin WT) and 18786 (Erbin S47).
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures and PyMOL session scenes
and can be found with this article online at http://dx.doi.org/10.1016/j.str.
2012.10.007.
ACKNOWLEDGMENTS
The authors thank Eli Groban for assistance in setting up kinase assays; Ian
Harwood and the lab of Robert Stroud for generously providing the modified
pET-47 expression vector, HRV-3C protease, and associated supplies; and
Julie Zorn, Justin Rettenmaier, and the lab of Jim Wells for invaluable help
with mass spectrometry analysis. This work was supported by an award
from the National Science Foundation (NSF) to T.K. (NSF CAREER MCB-
0744541) and the Synthetic Biology Engineering Research Center (SynBERC,
NSF-EEC-0540879). C.A. Smith was additionally supported by a DOD NDSEG
fellowship and the NSF GRFP. C.A. Shi, M.K.C., and T.E.B. were supported by
the UCSF summer research training program and SynBERC.
Received: July 18, 2012
Revised: October 13, 2012
Accepted: October 18, 2012
Published online: November 15, 2012
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