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1
Recognition of a functional peroxisome type 1 target by
the dynamic import receptor Pex5p
Will A. Stanley1, Fabian V. Filipp2, Petri Kursula1, Nicole Schüller1, Ralf
Erdmann3, Wolfgang Schliebs3, Michael Sattler2, Matthias Wilmanns1*
1EMBL-Hamburg Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany
2Structural and Computational Biology Unit, EMBL-Heidelberg, Meyerhofstrasse 1,
69117 Heidelberg, Germany
3 Institute for Physiological Chemistry, Department of Systems Biology, Faculty of
Medicine, Ruhr University of Bochum, 44780 Bochum, Germany.
*Correspondence:
Email: [email protected]
Phone: +49-40-89902-110
Fax: +49-40-89902-149
Running Title: Target recognition by the import receptor Pex5p
* C) Manuscript
2
Summary
Peroxisomes require the translocation of folded and functional target proteins of
various sizes across the peroxisomal membrane. We have investigated the
structure and function of the principal import receptor Pex5p, which recognizes
targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal
structures of the receptor in the presence and absence of a peroxisomal target,
sterol carrier protein 2, reveal major structural changes from an open, snail-like
conformation into a closed, circular conformation. These changes are caused by a
long loop C-terminal to the seven-fold repeated tetratricopeptide repeat segments.
Mutations in residues of this loop lead to defects in peroxisomal import in human
fibroblasts. The structure of the receptor/cargo complex demonstrates that the
primary receptor binding site of the cargo is structurally and topologically
autonomous, enabling the cargo to retain its native structure and function.
3
Introduction
Diverse machineries involved in translocating proteins across organellar membranes are
required to maintain the compartmentalization of biological processes within eukaryotic
cells (Kunau et al., 2001; Wickner and Schekman, 2005). Many components of
membrane receptors recognize specific targeting sequences in proteins destined for
translocation (Eichler and Irihimovitch, 2003). However, they differ in their
requirements for cargo folding/unfolding during the translocation process to retain the
functional integrity of the cargo. To date, the only well characterized system supporting
the translocation of folded protein targets is the nuclear import/export system by
karyopherins (Conti and Izaurralde, 2001; Matsuura and Stewart, 2004). The extent to
which other translocation systems may resemble karyopherin-mediated processes
remains elusive.
Peroxisomal import is one of the few transport processes that uses a translocon for the
purpose of trafficking folded and functional cargo proteins across membranes (Gould
and Collins, 2002; Holroyd and Erdmann, 2001; Lazarow, 2003; Schnell, 2000; van der
Klei and Veenhuis, 2002). To date, more than two dozen proteins involved in
trafficking cargo to the peroxisome—referred to as peroxins—have been identified and
partially characterized. No pore-like structure in the peroxisomal membrane has thus far
been observed, and the exact composition of the import translocon, possibly assembled
according to the size and type of import substrates, has yet to be established. The
majority of peroxisomal matrix proteins destined for translocation into peroxisomes
share the C-terminal type 1 peroxisomal targeting signal (PTS1) motif. It comprises an
obligatory C-terminal tripeptide, conforming to the consensus sequence -[S/A/C]-
[K/H/R]-[L/M]-CO2-, which is specifically recognized by the C-terminal segment of the
4
import receptor peroxin Pex5p. Human diseases leading to impaired fatty acid
metabolism, organ dysfunction, and neonatal mortality, such as Zellweger syndrome,
are often caused by mutations in the Pex5p receptor (Weller et al., 2003), rendering the
receptor an important target for biomedical research.
At the molecular level, these Pex5p translocation targets appear to be released into the
peroxisomal lumen by interactions between the receptor and other peroxins and by its
association with the peroxisomal membrane (Gouveia et al., 2003a; Holroyd and
Erdmann, 2001; Madrid et al., 2004). Cargo loading may also influence the oligomeric
state of Pex5p and its interactions with other peroxisomal membrane docking factors,
such as Pex14p (Madrid et al., 2004; Wang et al., 2003). Other evidence demonstrates
that the cargo-loaded Pex5p receptor may even shuttle across the peroxisomal
membrane (Dammai and Subramani, 2001). However, to date, questions still remain
regarding whether, or to what extent, the receptor, or parts of the receptor, physically
shuttle or just become accessible to the peroxisomal lumen (Erdmann and Schliebs,
2005; Kunau et al., 2001).
To investigate the molecular requirements of this dynamic receptor both for cargo
loading and release, we determined the structures of the PTS1-cargo binding region of
the Pex5p receptor in the presence and in the absence of a peroxisome translocation
target, sterol carrier protein 2 (SCP2). The target is bound to the receptor by two
separate binding sites—a C-terminal PTS1 motif and a topologically separate secondary
site—providing a rationale as to how the target remains folded and functional during
translocation. A comparative analysis of the two Pex5p receptor structures reveals major
conformational changes in the receptor upon cargo loading, which are generated by the
loose structural arrangement of the receptor tetratricopeptide repeat (TPR) segments and
5
by the intrinsic structural flexibility within some of these structural segments. Our data
reveal unexpected common principles governing the conformational changes observed
in the peroxisome Pex5p import receptor and karyopherins, providing insight into the
major determinants of protein import/export into the peroxisome.
6
Results
Choice of a functional receptor/cargo system
In order to unravel the molecular basis of PTS1-driven protein translocation via the
Pex5p receptor into peroxisomes, we searched for a suitable physiological target that
could serve as a model system. We selected sterol carrier protein 2 (SCP2), which
contains a canonical C-terminal PTS1 motif (Seedorf et al., 1998). Its structure has been
characterized previously, both by X-ray crystallography and NMR spectroscopy
(Choinowski et al., 2000; Garcia et al., 2000). The SCP2 gene is translated into two
protein products: SCPx, a 58 kDa fusion protein comprising an N-terminal thiolase
domain and a C-terminal SCP2 domain, and preSCP2, a protein with a molecular mass
of about 15 kDa, which is processed into its mature form (mSCP2) by proteolytic
cleavage of a 20-residue leader sequence after translocation into peroxisomes (Figure
1). SCP2 binds to the Pex5p receptor both under in vivo and in vitro conditions,
allowing structural investigation of the receptor/cargo complex.
We used NMR spectroscopy and isothermal titration microcalorimetry (ITC) to
determine the molecular requirements of SCP2 for receptor binding. For both the
translated (pre) and the processed mature (m) forms—preSCP2 and mSCP2,
respectively—the chemical shift perturbations in 1H,15N correlation spectra upon
binding to Pex5p(C) affect the same set of SCP2 residues (Supplement, Figures S1
and S2). Furthermore, chemical shift perturbation and line width analysis show that the
presequence remains highly dynamic and is not involved in receptor binding. The
binding affinities of preSCP2 and mSCP2 for the receptor, as measured by ITC, are
7
both in the order of 100 nM (Table 1), indicating that the presequence is tolerated and
does not affect the receptor interaction.
To determine whether SCP2 retains its function upon loading onto the Pex5p import
receptor, we used two different but complementary approaches. First, we investigated
the capacity of SCP2 to bind specific lipids required for metabolic processes inside
peroxisomes in the presence and in the absence of Pex5p(C). To locate the lipid binding
site with SCP2, we used spin-labeled paramagnetic doxyl stearate as a fatty acid
derivative (Garcia et al., 2000). We observed similar bleaching of NMR signals upon
binding of this fatty acid derivative to either free SCP2 or Pex5p(C)-bound SCP2,
indicating that the functional integrity of SCP2, in terms of lipid binding, is not
impaired upon receptor loading (Figure 2). We further used ITC to quantify the
thermodynamic parameters governing the receptor-cargo interaction in the presence and
in the absence of stearoyl-CoA, a physiological ligand of SCP2 (Frolov et al., 1996).
The data show that mSCP2 binds to the receptor with about the same Kd and 1:1
stoichiometry, regardless of whether it is loaded with stearoyl CoA (Table 1).
Structure of the cargo-loaded Pex5p receptor complex
We have determined the crystal structure of the C-terminal part of the Pex5p import
receptor (Pex5p(C), residues 315-639) in the presence of the mature form of the
peroxisomal matrix protein SCP2, mSCP2(21-143), at 2.3 Å resolution (Figures 1, 3A-
B, 4A-B, 5; Table 2). The X-ray data reveal the complete structure, except for the very
N-terminus (315-334) and one loop (441-453) of Pex5p(C). Most of its structure is
formed by seven consecutive TPR motifs, each consisting of a helix-turn-helix motif
(D'Andrea and Regan, 2003). The forth segment, which matches the established TPR
8
sequence signature, is in a distorted arrangement (Figure 5A) and is preceded by a
glycine-rich loop that is, in part, flexible. The C-terminus of the structure is folded into
a three-helical bundle of which the first two helices display TPR-like properties, both in
terms of sequence signature pattern and structure. The long loop connecting the seventh
TPR segment and the C-terminus (589-601), referred to as “7C-loop”, interacts with the
two helices from the TPR1 segment (Figure 5B). This loop and the distorted TPR4
segment link the two arch-shaped TPR motif triplets (1-3, 5-7), thus generating a
pseudo-circular structure of the cargo-loaded receptor with a tunnel in its center, which
is open to both faces of the ring-like structure (Figure 4B). The connecting segments
represent the most mobile regions of the cargo-loaded receptor (Supplement, Figure
S3A) and, therefore, can be regarded as hinges. The C-terminal helical bundle does not
participate in the circular arrangement (Figures 3A-B and 5A-B).
The structure of the receptor-bound mSCP2 resembles that of free SCP2 (Choinowski et
al., 2000), except the C-terminus that bears the PTS1 motif (Supplement, Figure S3C).
In the Pex5p(C) receptor complex, the ten C-terminal residues (134-143) of mSCP2
adopt an extended conformation, pointing away from its core domain. However, unlike
the structure of free SCP2, wherein both termini are flexible, in the receptor-bound
SCP2 structure, the C-terminus becomes the most rigid part of the overall structure
while its N-terminus remains mobile (Supplement, Figures 1C and S3B-C). The most
C-terminal AKL motif (141-143) of mSCP2 binds within the central hole of the ring-
like structure of the receptor. It is involved in specific interactions with four asparagines
(N415, N526, N534, N561) that are located on the N-terminal helices of TPR segments
4, 5, 6, and 7. These residues are conserved (Figures 1, 5A-B), indicating that PTS1
binding is a general property of the Pex5p receptor. In contrast to the most C-terminal
9
region, the preceding residues (134-140) interact with the receptor by van der Waals
forces only.
The structure of the receptor/cargo complex also reveals a second interaction site of
about 500 Å2 that is formed by the C-terminal helical bundle of Pex5p(C) and a surface
patch of SCP2, covering parts of helices 1 and 3 (Figures 1, 4A-B, 5B). ITC binding
data using the PTS1 peptide motif only reveal that its binding affinity to Pex5p(C) is
reduced (Kd = 664 nM) compared with the entire protein cargo (Table 1). Thus, the data
indicate that there is a notable contribution by the secondary interface in SCP2 loading
onto the Pex5p receptor. However, none of the residues of either the Pex5p receptor or
SCP2 involved in these interactions are invariant (Figure 1). These findings suggest
that, in contrast to the PTS1-mediated cargo/receptor interactions, the formation of the
secondary SCP2/Pex5p(C) interface is specific and may not be conserved taxonomally.
Since PTS1 targets are generally unrelated in terms of structure and function, with the
exception of the C-terminal PTS1-receptor recognition motif, our findings suggest that
the involvement of secondary binding sites may serve as a determinant for the sorting of
folded import substrates.
Our NMR spectroscopy data on mSCP2 in the presence and in the absence of the
receptor correlate with the crystallographic analysis. The largest chemical shift
perturbations are found for the backbone amides of the C-terminus (136-143)
(Supplement, Figure S1B), coinciding with the structural alterations observed by
comparing the structures of unbound SCP2 and Pex5p(C)-bound SCP2 (Figure 5B;
Supplement, Figure 3C). NMR relaxation measurements indicate that the PTS1
backbone, which is flexible and disordered in free SCP2, rigidifies upon binding to
10
Pex5p(C) (Supplement, Figure 1C). Significant chemical shift changes have also been
detected for residues in the secondary binding site.
Taken together, our biochemical and structural data suggest bipartite binding of the
SCP2 cargo to the Pex5p receptor. The PTS1 binding site at the most C-terminal region
is structurally and topologically well separated from the functional SCP2 core domain.
As such, the data provide a rationale for our observations on a mechanism of receptor
recognition that does not interfere with the function of SCP2 as lipid binding protein.
Our data, however, do not support previous hypotheses proposing that binding of lipid
substrates to members of the SCP2 family may enhance the exposure of the PTS1 motif,
thereby driving ligand-dependent translocation (Choinowski et al., 2000; Garcia et al.,
2000; Lensink et al., 2002).
Structure of the unliganded Pex5p receptor
Protein translocation into peroxisomes requires a delicate balance between the binding
and release of cargo proteins to and from the appropriate import receptor. To investigate
the molecular parameters that govern cargo release, we have also determined the
structure of the import receptor Pex5(C) in the absence of a cargo at 2.5 Å resolution
(Figures 3C, 4C; Table 2). We have found a crystal form that contains four Pex5p(C)
molecules per asymmetric unit. For two of these, the entire sequence is well defined in
electron density, except for a gap from part of the TPR4 segment. In the other two
Pex5p(C) molecules, the TPR segments 5-7 and the C-terminal helical bundle are
reasonably well defined, whereas the N-terminal TPR domains 1-3 could only be
modeled approximately, as reflected in high average mobility factors (Table 2,
Supplement Figure S3A). There is, however, no evidence to support the unfolding of
11
considerable parts of the TPR segments. Previous circular dichroism data have shown
that the secondary structural content of both receptor conformations is almost identical
(Stanley et al., 2004).
A direct comparative analysis of the cargo-loaded and unloaded structures of the
Pex5p(C) receptor reveals major conformational changes upon cargo binding. Contrary
to previous hypotheses proposing that the TPR4 segment is a flexible hinge (Gatto et
al., 2000), these changes originate from three residue clusters in TPR segments 5 and 6,
rendering a rotation of about 20 degrees of the C-terminal TPR segments with respect to
N-terminal TPR segments (Figure 3D). As a result of this conformational change, the
7C-loop of the apo-structure is no longer capable of completing a ring-like structure of
Pex5p(C) as observed in the cargo complex, thus generating an open, snail-like
arrangement of the receptor (Figure 3C, Figure 4C). For instance, Gln586 and Ser600,
which interact with residues from the TPR1 segment in the cargo-loaded receptor
(Figure 5B), have moved by more than 8 Å in the apo-structure.
Since the overall arrangement of TPR segment–containing structures can be described
as a superhelical coil or solenoid (D'Andrea and Regan, 2003; Jinek et al., 2004), we
compared the underlying structural parameters of the cargo-free and cargo-loaded
structures of the Pex5p receptor. Our analysis revealed that the superhelical pitch is
about 30 Å in the apo-structure rather than 20 Å in the SCP2-Pex5p complex.
Furthermore, the N-terminal helices of TPR7 (556-568) and the C-terminal helical
bundle (601-613), which bear several residues that are involved in binding of the cargo
PTS1-motif, are moved by the equivalent of about two -helical pitches with respect to
the N-terminal TPR segments, leading to a displacement of part of the PTS1 motif
binding site by several Ångstroms (Figure 3D). The highly flexible arrangement of the
12
third and the fourth molecule found in the apo-Pex5p(C) crystals suggests that there
may be even more conformational freedom than that observed in the available apo-
crystal structures. These suggestions are consistent with our ITC data, indicating that
although the binding affinity is in the nanomolar range, there is only a small
contribution, or even a loss of entropy, during cargo binding (Table 1). Indeed, a recent
study has demonstrated coupled folding and ligand binding in at least one TPR array
(Cliff et al., 2005).
Our model allows for speculation on further possible structural changes. Although
TPR4 is not involved in the conformational changes evident from our comparative
analysis of the apo-and cargo-loaded Pex5p receptor structures, we cannot exclude the
possibility that there are steps during the target-import cycle that affect this receptor
segment as well. For instance, complete folding of the distorted TPR4 motif into the
canonical TPR domain structure would only require minor changes in the flexible loop
N-terminal to the TPR4 segment (Figure 5A). The resulting overall structure could
open up into a superhelical arrangement with a pitch in the order of 55 Å, reminiscent of
previous observations in another TPR segment–containing structure (Jinek et al., 2004).
In contrast to the observed changes in the receptor structures in the presence and in the
absence of cargo, the overall conformation of the cargo-loaded Pex5p(C) receptor
structure remains virtually identical regardless of whether it is bound only to the C-
terminal PTS motif (Gatto et al., 2000) or to a complete cargo target, as shown by the
Pex5p-SCP2 complex.
Residues from the 7C-loop are critical for in vivo PTS1 import
In contrast to several known peroxisome disease mutations wherein direct interactions
13
with the PTS1 motif are abolished, a patient with an inherited peroxisome biogenesis
disorder, infantile Refsum disease, was found to be impaired in the import of proteins
containing only the AKL- and KANL-type PTS1 motifs, such as SCP2 and catalase
(Shimozawa et al., 1999). In this patient, dysfunctional import into peroxisomes is
linked to mutation S600W in Pex5p. Comparative analysis of the cargo-loaded and
unloaded structures of the Pex5p(C) receptor reveals that Ser600, which is situated at
the base of the 7C-loop, plays a central role in connecting the C-terminal and N-
terminal TPR segments, to arrange the PTS1 binding site as well as the secondary
binding site at the C-terminal helical bundle in the Pex5p(C)/SCP2 complex (Figure
5B).
In order to validate the involvement of the 7C-loop in PTS1 target import we mutated
three residues (Gln586, Ser589, Ser600), which are involved in specific interactions of
this loop with other parts of the receptor in the cargo-bound bound conformation
(Figure 5B). As a control, we chose one single residue mutant from the TPR2 segment
(N382A), which previously was shown to be involved in PTS1 cargo import by the S.
cerevisiae Pex5p receptor (Klein et al., 2001). First, we measured the binding affinity of
the cargo SCP2 to the resulting Pex5p variants under in vitro conditions (Table 1). As
expected, no binding could be detected for the S600W mutant, while a more than ten-
fold reduced binding affinity was observed for the Q586R Pex5p variant, thus
demonstrating the critical involvement of this residue from the 7C-loop in SCP2 cargo
recognition as well. On the other hand, the cargo binding by the Pex5p S589Y was only
slightly reduced.
The same mutations were introduced in full-length Pex5p and the resulting variants
were expressed in a fibroblast cell line devoid of endogeneous PTS1 receptor.
14
Transfected cells were analyzed by fluorescence microscopy for their capacity to import
two established PTS1 peroxisome targets, catalase and SCP2 (Shimozawa et al., 1999).
While SCP2, due to its low molecular weight and absence of evidenced tendencies for
oligomerization, can be considered as a model target with only modest structural
requirements for import in its functional form, catalase forms a homo-tetrameric heme
containing assembly with a molecular mass of about 240 kDa, thereby demanding
considerable additional structural requirements for peroxisomal import (Purdue and
Lazarow, 1996). As a control, we tested Pex5p-dependent import of the PTS2-tagged
reporter protein, chloramphenicol acetyltransferase (CAcT) as well. To ensure that the
observed import defects are not because of a reduced amount of mutant due to lower
expression or increased turnover we have analyzed the expression of all Pex5p mutants
in a Pex5p-free cell line 24 hours after transfection with the corresponding plasmids by
immunoblotting (Supplement, Figure S4), demonstrating that all Pex5p mutants were
synthesized at their full-length. All mutant proteins are found at a steady-state level
comparable with wt Pex5p expressed from the same plasmid. The levels of expression
were significantly increased when compared with wt Pex5p under control of its
endogenous promotor.
The transfected wt Pex5p receptor correctly directed both PTS1 targets, catalase and
SCP2, as well as the PTS2-tagged CAcT into the peroxisomal matrix (Figure 6). As
expected, two of the 7C-loop mutants (Q586R, S589Y) and the PTS1 reference mutant
(N382A) mediated properly the import of PTS2 proteins but showed severe defects for
catalase targeting. Retarded import was observed for SCP2, most apparently when
expressing the Q586R mutant. During the first 24 hours after transfection the bulk of
GFP-SCP2 remained in the cytosol and only a few peroxisomes were detected by
fluorescence (Figure 6). The number of SCP2 containing peroxisomes further increased
15
with incubation time and after two to four days nearly all peroxisomes were labeled
(data not shown). Moreover, the Pex5p S600W mutant was not able to rescue either
catalase or SCP2 import during the whole time-course of the experiment (Figure 6).
By taking the our in vitro and in vivo data together, both lines of evidence demonstrate
the essential contribution of the Ser600 side chain in the peroxisomal import of PTS1
targets by the Pex5p receptor. Indeed, Ser600 is the only residue at the distal end of the
7C-loop that connects the C-terminal helical bundle with the TPR segments via a
specific hydrogen bond to a residue from the TPR1 motif, upon SCP2 cargo binding. In
structural terms, abolition of this interaction is expected to impair both the function of
the 7C-loop as closing element of the ring-like conformation of the cargo-bound
receptor as well as on the proper arrangement of the C-terminal helical bundle with
respect to the N-terminal TPR segments of the receptor. In contrast, the other two
mutants (Q586R, S589Y) are located at the N-terminus of the 7C-loop, which is
proximal to the preceding TPR segments. Gln586 is involved into a multi-hydrogen
bond network, involving residues from the first TPR segment and Ser600 (Figure 5B).
Our data show that abolition of specific interactions from these residues, in particular of
Gln586, leads to serious import defects as well. Comparison of the data for SCP2 and
catalase, however, indicates an amplification of the effect for the latter one, which is
expected to be more sensitive because of its oligomeric arrangement and requirement
for cofactor binding. Although a quantitative interpretation of this differential effect will
only be possible once a structure of receptor-catalase cargo complex becomes available,
it is intriguing to hypothesize on additional sorting effects for the import of different
PTS1 cargos, supporting or complementing previous observations (Kiel et al., 2004;
Knott et al., 2000; Otera et al., 2002).
16
Discussion
Pex5p receptor recognition of diverse PTS1 targets
We have analyzed our structural findings of the Pex5p import receptor to determine
whether, or to what extent, they infer general principles applicable for the translocation
of a variety of folded proteins into peroxisomes. For mSCP2, our data suggest a
bipartite recognition mechanism, via the C-terminal PTS1 motif and a secondary, less
conserved binding site that is distinct in terms of sequence and structure. Much attention
has previously focused on residues preceding the C-terminal tripeptide PTS1 motif,
suggesting that some of these may serve as determinants for altering binding affinity
(Lametschwandtner et al., 1998; Neuberger et al., 2003). However, the structure of the
Pex5p(C)/mSCP2 complex has not revealed any further specific cargo interactions with
the receptor beyond the C-terminal PTS1 motif. Although several hydrophobic
interactions can be observed these interactions may change in other PTS1 targets, in
which the orientation of the linker region between the PTS1 motif and the functional
domain structures may be different. As such, it is difficult to quantify possible
contributions of residues from the linker, connecting the core domain of SCP2 and the
PTS1 C-terminus to Pex5p receptor.
Recent data on the recognition of another peroxisome target, alanine:glyoxalate
aminotransferase (AGA), by the Pex5p receptor indeed support our findings on bipartite
cargo binding by a second topologically remote interaction site separated by about 40
residues in the AGA sequence (Huber et al., 2005). Since considerable variation has
been observed in the sequence within the PTS1 motif of different targets, it is plausible
17
to assume that there is a correlation between a weakened PTS1-Pex5p receptor
interaction and a need for secondary binding sites on the cargo surface.
A peculiar property of the Pex5p import system is its capability to allow translocation of
even large-folded proteins, either monomeric or oligomeric (Brocard et al., 2003;
Lazarow, 2003; Walton et al., 1995; Yang et al., 2001). Some PTS1 targets can be
imported into peroxisomes either by a PTS1-dependent or PTS1-independent
mechanism (Parkes et al., 2003). On the other hand, there has been recent biochemical
and structural evidence demonstrating that oligomerization of proteins established as
physiological peroxisomal targets may actually inhibit PTS1-driven translocation (Faber
et al., 2002; Modis et al., 1998). In this scenario, a possible blockade of secondary
binding sites, as, for instance, by oligomerization, appears to be more likely than the
PTS1 motif, as predicted by previous data (Parkes et al., 2003) and supported by the
Pex5p(C)/mSCP2 complex structure reported here.
The Pex5p import receptor cycle
The generally accepted model for PTS1-driven import of peroxisomal targets considers
at least four steps for one import cycle: (a) cargo recognition by the Pex5p receptor, (b)
cargo-loaded receptor docking and, possibly, integration into the peroxisomal
membrane, (c) cargo release into the peroxisomal lumen, and (d) recycling of the
unloaded receptor into the cytosol (Erdmann and Schliebs, 2005). The model proposes
two essential types of binding/unbinding events that are likely to be associated with
considerable conformational changes in the receptor: loading/unloading of the cargo and
docking/release of the receptor to/from the peroxisomal membrane. For canonical
PTS1-driven import of peroxisomal targets, loading/unloading of the cargo seems to be
18
confined to the C-terminal part of the receptor. Our comparative analysis of the near
atomic resolution structures of the Pex5p(C) receptor enables the unraveling of the
molecular parameters governing the conformational changes associated with cargo
loading. In contrast, data on the N-terminal part of Pex5p, which is thought to be largely
unfolded (Costa-Rodrigues et al., 2005), have indicated that this region is critically
involved in membrane docking/release of the receptor, either by interactions with other
docking factors of the translocon, such as Pex13p and Pex14p as well as Pex8p and
Pex17p (only demonstrated in yeast), or by direct interactions with the peroxisomal
membrane (Agne et al., 2003; Gouveia et al., 2003b; Schäfer et al., 2004). The model
implies that these interactions could have an effect on the affinity of the peroxisomal
target for the C-terminal part of the receptor, ultimately leading to cargo release. Pex8p
appears to play a key role in this process, either as a cargo release factor, as suggested
from in vitro data of the H. polymorpha Pex5p receptor (Wang et al., 2003) or as an
inducer of a subsequent translocon complex with additional components allowing cargo
release (Agne et al., 2003). Regardless the precise origin of the conformational changes
in the C-terminal part of the receptor, both the canonical nature of the loose
arrangement of the TPR domains and our direct comparative analysis of the cargo-
loaded and unloaded receptor structures suggest a ring-opening mechanism for
peroxisomal target release.
Common principles in protein translocation systems
A direct comparative analysis of the apo- and cargo-loaded conformations of the Pex5p
receptor has led to unraveling the molecular basis of loading the most widespread class
of peroxisomal targets—i.e. those containing a C-terminal PTS1 signal motif—onto the
receptor for import into peroxisomes. However, data from other peroxisome receptors
19
are too scarce to allow further investigations of common/divergent principles involved
in peroxisomal import. To date, the most widely investigated family of receptors have
been the karyopherins, which are involved in the signal-assembled import/export of a
wide range of targets into the nucleus (Chook and Blobel, 2001; Conti and Izaurralde,
2001). These receptors recognize nuclear localization signal (NLS) containing targets
either directly or via adaptor proteins, with cargo loading/unloading being regulated by
binding to RanGDP/GTP binding proteins. Unlike the case for nuclear transport, for
PTS-driven translocation by the Pex5p receptor, there is no evidence supporting
protein-mediated cargo translocation or involvement of further loading factors.
Nevertheless, the general principles for protein translocation inferred from our data are
surprisingly similar to those governing karyopherins. First, NLS peptides are bound in
an elongated coiled conformation, as is found here with PTS1. In addition, large parts of
karyopherin receptors comprise repeated -helical domain structures that are classified
by HEAT or ARM sequence motifs, which are analogous to those in the TPR segments
of the Pex5p receptor. In terms of overall arrangement of receptor recognition, our
findings on the cargo-Pex5p receptor complex are, for instance, reminiscent of the
structure of the cargo-loaded Kap()1 receptor, in which the circular structure of the
receptor binds an N-terminal fragment of the Kap() substrate adaptor into a central
tunnel-like cavity (Cingolani et al., 1999). Furthermore, recently determined structures
of the export receptor Cse1 in the apo-conformation (Cook et al., 2005), RanGTP-
bound form (Lee et al., 2005) as well as the ternary complex with RanGTP and
Kap60p (Matsuura and Stewart, 2004) have revealed unexpected structural insight into
the molecular parameters that support binding of the Kap60p adaptor, which, in turn,
inhibits binding of the NLS-containing targets. Comparison of these data with the
cargo-loaded/unloaded conformations of the Pex5p receptor reveal two further common
20
principles governing structural adaptations during the respective translocation cycles. In
both systems, overall conformational changes are triggered by local changes in
structural modules that may be propagated into modified arrangements with adjacent
modules. For instance, a comparison of the cargo-loaded structure with the unloaded
structure of karyopherin Cse1 have unraveled a conformational switch in the HEAT
repeat 8 (Conti and Izaurralde, 2001; Cook et al., 2005), leading to altered HEAT repeat
arrangements N- and C-terminal to the switch. Taken together, the comparative analysis
of the cargo-loaded and unloaded structures of the Pex5p receptor provides evidence for
a similar structural role of the TPR4 segment and adjacent TPR regions. Second, in both
systems—the karyopherins and the Pex5p receptor—conformational changes can be
described by alterations in the underlying geometrical parameters of each superhelical
domain arrangement. In both systems, binding of regulators, adaptors, and/or substrates
lead to changes in the overall superhelical pitch values that may be described as ring-
like, snail-type, and open arrangements (Conti and Izaurralde, 2001; Lee et al., 2005).
Thus, emerging evidence points to structural and functional similarities in the molecular
principles governing unrelated translocation receptors containing repeated helical
domain structures. Indeed, in the light of the recently detected common architectural
features of protein components of nuclear pore complexes and coated vesicles (Devos et
al., 2004; Devos et al., 2006) it may well be possible that there are common principles
in structural rearrangements in an increasing list of proteins with -solenoid
conformations, in which our findings on structural plasticity of the Pex5p import
receptor fit as well.
21
Experimental Procedures
Protein preparation
All versions of human Pex5p(C) (residues 315-639), human preSCP2 (residues 1-143),
and mSCP2 (residues 21-143) were expressed from a modified pET24d vector
(prepared by G. Stier, EMBL-Heidelberg) in E. coli BL21(DE3). Mutants N382A,
Q586R, S589Y and S600W of Pex5p(C) were created using the Quickchange XL-Site
Directed Mutagenesis Kit (Stratagene). The expressed proteins contained an N-terminal
His6-GST fusion, cleavable with tobacco etch virus (TEV) protease. Cultures were
grown in Tris-buffered LB medium supplemented with 1% (w/v) glucose, and induced
mid-log-phase with 0.5 mM IPTG for 6 hours at 21 °C. Following resuspension of the
cell pellets, cells were lysed by sonication in the presence of protease inhibitors. The
lysate was cleared by centrifugation, loaded onto a glutathione Sepharose 4B resin
(Pharmacia), and then eluted with 20 mM reduced glutathione. TEV protease was added
until fusion proteins were completely cleaved and the mixture was applied to Ni-NTA
agarose (QIAgen). The flow-through was subsequently subjected to gel filtration
through a Superdex 75 (16/60) column (Pharmacia).
Crystallization and X-ray data acquisition
Pex5p(C) and mSCP2 were mixed in a 2:3 molar ratio and dialyzed against 20 mM bis-
Tris-propane, 20 mM KCl, and 1 mM TCEP (pH 7.0). The protein mixture was then
concentrated to ~7 mg ml-1 total by ultrafiltration, and crystallization was carried out by
mixing 1 l protein with 1 l reservoir solution using the hanging drop vapor diffusion
method at 20 °C. Reservoir buffer conditions were optimized to 24% (w/v) PEG 3350,
22
175 mM NaCl and 100 mM bis-Tris (pH 6.5). Crystals grew within 4-6 weeks. Prior to
X-ray data collection, 10% (v/v) PEG 400 was added to the drops containing crystals
for 5-10 mins. Crystals of unliganded Pex5p(C) were obtained by mixing 1 l protein at
a concentration of 7 mg/ml with 1 l reservoir solution using the hanging drop vapor
diffusion method at 20 °C. Reservoir buffer conditions were optimized to 23% (w/v)
PEG 3350, 100 mM Tris-HCl (pH 8.75), and 0.22 mM octaethylene glycol monolauryl
ether. Crystals grew within 3-5 weeks.
X-ray data were collected at the synchrotron beamlines X13 at EMBL/DESY,
Hamburg, Germany, and at BL1 at BESSY, Berlin, Germany. Data were obtained from
single crystals at 100 K in a stream of gaseous nitrogen. Data were processed and scaled
using XDS (Kabsch, 1988), and then analyzed using SFCHECK (Vaguine AA et al.,
1999). For cross-validation of subsequent steps, 5% of the reflections from each data set
were randomly selected.
X-ray structure determination
Pex5p(C)/mSCP2 complex: The X-ray structure was solved by molecular replacement
using MOLREP (Vagin and Teplyakov, 1997). Initially, Pex5p(C) was located using the
PDB coordinates 1FCH (Gatto et al., 2000) as a model. Subsequently, SCP2 was found
using the PDB coordinates 1C44 (Choinowski et al., 2000) as a model. REFMAC-5
(Murshudov, 1997) was used to refine the structure, applying TLS parameterization
(Winn et al., 2001). Simulated annealing refinement was carried out in CNS (Bruenger
et al., 1998). Manual building and structure analysis were carried out in O (Jones et al.,
1991). Solvent molecules were constantly added both manually and by ARP/wARP
(Lamzin and Wilson, 1993). The structure quality was assessed using PROCHECK
23
(Laskowski et al., 1993). Residues 335-440 and 454-639 of Pex5p and residues 22-143
of SCP2 were included in the final model.
Unliganded Pex5p(C): In order to find a molecular replacement solution using the
program MOLREP (1994; Vagin and Teplyakov, 1997), the Pex5p coordinates of the
SCP2/Pex5p complex structure were split into two parts, spanning residues 335-440 and
454-637. All four copies of the C-terminal part, but only two copies of the N-terminal
part could be identified. Refinement of these molecule fragments was carried out using
REFMAC5 (Murshudov, 1997). The program O (Jones et al., 1991) was used for model
building and analysis. Analysis of the electron density after initial refinement and
rebuilding indicated weak electron density for the two missing N-terminal molecule
fragments, allowing the determination of the overall orientation of each domain based
on the densities of the known helices. The orientation of each N-terminal part relative to
the C-terminal part was essentially the same as in the two well defined molecules.
Refinement was continued by applying NCS restraints separately to the N- and C-
terminal halves of the four Pex5p monomers. Furthermore, the TLS refinement option
in REFMAC5 was used by defining each monomer as a single rigid body. Due to the
high amount of structural flexibility in the N-terminal parts of two of the four Pex5p
molecules in the crystal and the anisotropy of the X-ray diffraction data, the final R-
factors remained higher than those for the Pex5p/cargo complex. Programs of the CCP4
package (Collaborative Computational Project, Number 4, (1994) were also used for
structure manipulation, analysis, and validation.
NMR spectroscopy
24
Isotopically-labeled (90% 2H, 13C and/or 15N) SCP2 was prepared by growing bacteria
in minimal medium supplemented with [U-13C] glucose and/or 15NH4Cl in D2O.
Proteins/complexes were exchanged into 100 mM potassium phosphate (pH 6.5) by gel
filtration. Samples were used at concentrations of 0.2-1.0 mM. NMR spectra were
acquired at 22 °C (complex) or 37 °C (free SCP2) on Bruker spectrometers (DRX600
with cryogenic probe or DRX900 with triple resonance probe). The backbone chemical
shifts of preSCP2 and mSCP2 were based on BMRB entry 4438 (Garcia et al., 2000)
and extended using standard triple resonance experiments (Sattler M et al., 1999). The
assignments for SCP2 in the 50 kDa SCP2/Pex5p(C) complexes were obtained using
triple resonance and 15N-edited TROSY-NOESY experiments on samples comprising
2H,15N- or 2H,13C,15N-labeled SCP2 and unlabeled Pex5p(C). Chemical shift
perturbations ( = [(1H)2 + (1/5 15N)2]½ , in parts per million) were monitored in
two-dimensional 1H,15N-TROSY experiments. T1 15N relaxation was measured at 600
MHz with a spin-lock field strength of 2 kHz. Spin-label induced paramagnetic
relaxation enhancements were analyzed from intensity changes in 1H,15N-TROSY
experiments of SCP2 or SCP2/Pex5p(C) recorded in the presence of the fatty acid
derivative 5-doxyl stearic acid in the oxidized form and after reduction with ascorbic
acid (Battiste and Wagner, 2000).
Isothermal titration microcalorimetry
Proteins were co-dialyzed against 100 mM potassium phosphate (pH 7.4), 1 mM DTT.
SCP2 was pre-mixed with stearoyl-CoA (Sigma) in a small molar excess, and 2 M
stearoyl-CoA was added to the dialysis buffer to ensure uniform loading of SCP2.
Dialysates were degassed and the concentration measured by A280nm. ITC measurements
were conducted on a MicroCal VP-ITC using Pex5p(C) at 30-50 M as a sample and
25
SCP2 or PTS1 peptide (Sigma Genosys) at 350-750 M as the titration ligand.
Experiments were conducted at 35 °C using injection protocols found to saturate
Pex5p(C) with ligand. Ligand heats of dilution were subtracted and data fitted using
MicroCal Origin 5.0.
In vivo peroxisome import assays
Point mutations were introduced into the Pex5p sequence by using the Quickchange XL
– Site Directed Mutagenesis Kit (Stratagene). The pcDNA3 derived expression vector
pGD106 (Braverman et al., 1998) was used as a template to replace glutamine 586 by
arginine (Q586R; sense-primer: 5´-
GAGGCCCTGAACATGAGGAGGAAAAGCCGGGG-3´; antisense-primer: 5´-
CCCCGGCTTTTCCTCCTCATGTTCAGGGCCTC-3´), serine 589 by tyrosine
(S589Y; sense-primer: 5´-AACATGCAGAGGAAATACCGGGGCCCCCGGGG-3´;
antisense-primer: 5´-CCCCGGGGGCCCCGGTATTTCCTCTGCATGTT-3´), serine
600 by tryptophane (S600W; sense-primer: 5´-
GGAGGTGCCATGTGGGAGAACATCTGG-3´; antisense-primer: 5´-
CCAGATGTTCTCCCACATGGCACCTCC-3´) and asparagine 382 by alanin (N382A;
sense-primer: 5´-GTACCACCCAGGCAGAGGCTGAACAAGAACTATTAG-3´;
antisense-primer: 5´-CTAATAGTTCTTGTTCAGCCTCTGCCTGGGTGGTAC-3´).
mSCP2 was amplified by PCR using E.coli mSCP2 expression plasmid as a template
and the primers 5´-GATCTCGAGCCATGGGCTCTGCAAGTG-3´ and 5´-
TGAATTCAGAGCTTAGCGTTGCCTG-3´. The resulting fragment was digested with
restriction endonucleases XhoI/EcoRI and subcloned into the corresponding sites of
pEGFP-C1 plasmid (Clontech Laboratories, Inc.) thereby generating the expression
construct for the fusion protein EGFP-SCP2.
26
pPTS2-CAcT encoding an N-terminal PTS2 signal followed by the reporter protein
chloramphenicol acetyltransferase (CAcT) was described previously (Braverman et al.,
1998).
Human fibroblast cells were cultured at 37°C in Dulbecco modified Eagle’s medium
supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100.000 U/l penicillin,
and 100 mg/l streptomycin at 8% CO2. The human Pex5p-deficient skin fibroblast cell
line PBD005 (Dodt et al., 1995) was grown for one day on cover-slides in 60 mm
tissue-culture dishes before it was transfected with pPTS2-CAcT, pEGFP-SCP2 and one
of the Pex5p expression plasmids. Transfection was performed with 1-2 µg of each
plasmid DNA and 12 µl FuGENE 6 Transfection Reagent according to the
manufacturer’s instruction (Roche Diagnostics, Mannheim, Germany). At various time
points (24, 48, 72, 96 hours after transfection) cells were fixed on cover glasses with 3%
formaldehyde in phosphate-buffered saline (PBS), permeabilized with 1% Triton X-100
in PBS, and subjected to immunofluorescence microscopy and GFP life imaging.
Polyclonal rabbit antibodies against CAcT and sheep antibodies against human catalase
were purchased from Invitrogen (Germany) and The Binding Site (UK), respectively.
Secondary antibodies were conjugated with Alexa Fluor 594 or 488 (Invitrogen,
Germany).
All micrographs were recorded on a Zeiss Axioplan 2 microscope with a Zeiss Plan-
Apochromat 63x/1.4 oil objective and an Axiocam MR digital camera and were
processed with AxioVision 4.2 software (Zeiss, Jena, Germany).
Acknowledgments
27
We thank Ben Distel, André Klein, Ash Verma, Areti Malapetsas, Gunter Stier,
Christian Edlich, Christiane Sprenger, Elisabeth Becker, Bernd Simon and Elena Conti
for stimulating discussions and valuable support. This work was supported by the grants
HPRN-CT-2002-00252 (to M.W.) and LSHG-CT-2004-512018 (to R.E.) from the
European Commission, and grants Schl 584/1-1 and 1-2 (to W. Sch.) from the Deutsche
Forschungsgemeinschaft (DFG). We thank the DFG and the center for biomagnetic
resonance (BMRZ), Frankfurt, Germany, for access to the 900 MHz NMR instrument,
and BESSY, Berlin, Germany, for access to the synchrotron radiation beamline BL1.
Accession Numbers
Coordinates and structure factors have been deposited at the Protein Data
Bank with accession codes 2C0L and 2C0M .
28
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peroxisomes: either its own or that of a partner, Dci1p. Eur J Cell Biol 80, 126-
138.
36
Tables
Table 1: Thermodynamic characterization of Pex5p interaction with PTS1-
containing ligands by ITC.
Pex5p receptor CargoH
(kJ/mol)
TS
(kJ/mol)
G
(kJ/mol)
Kd
(nM)
wild-type mSCP2 -42.4 -1.2 -41.2 109 ± 34
wild-type mSCP2(SCoA) -31.8 8.9 -40.8 124 ± 17
wild-type preSCP2 -35.9 6.2 -42.1 74 ± 9
wild-type PGNAKL -45.1 -8.7 -36.4 664 ± 37
N382A mSCP2 -27.3 10.8 -38.1 348 ± 54
Q586R mSCP2 -17.4 17.3 -34.5 1343 ± 321
S589Y mSCP2 -38.7 1.20 -39.9 173 ± 23
S600W mSCP2 no binding
Table 1 legend:
SCoA, stearoyl coenzyme A. The measured stoichiometries deviated less than 10%
from a 1:1 complex, except for the Pex5p (S600W) mutant. Because of the experimental
errors in protein concentration measurements the stoichiometry values were adjusted to
1.00.
37
Table 2: Crystallographic statistics
Pex5p(C):mSCP2 Pex5p(C)
X-ray data collection statistics
Space group P212121 P1
Unit cell dimensions [Å] 40.5, 68.6, 137.4 53.5, 85.6, 88.9, 71.2°, 90.0°, 73.4°
Resolution range [Å] 25.0-2.3 (2.4-2.3) 20.0-2.5 (2.6-2.5)
Rsym [%] 9.4 (49.8) 13.7 (53.8)
I/σI) 14.1 (3.8) 6.3 (1.7)
Completeness [%] 99.8 (100.0) 95.9 (85.9)
Data redundancy 6.0 (6.1) 2.2 (2.1)
Unique reflections 17,692 47,257
Refinement statistics
Resolution range [Å] 20.0-2.3 20.0-2.5
R-factor/R-free [%] 20.2/25.6 26.3/30.9
Protein atoms 3209 9483
Solvent atoms 99 147
Rmsd bond distances [Å] 0.006 0.013
Rmsd bond angles [°] 1.0 1.4
Average B factors [Å2]a
Pex5p(C) N/Cb 18/30 14, 23,76,76/15,19,41,42
SCP2 39 -
Solvent 24 15
Rmsd B factors of protein bonded atoms [Å2]
38
Main chain 2.2 0.5
Side chain 2.6 1.1
Ramachandran plot regions [%]
Most favored 89.1 88.3
Additional allowed 10.0 10.6
Generously allowed 0.6 1.0
Disallowed 0.3 0.1
Table 2 Legend:
a TLS refinement parameters have been applied.
b N, Pex5p(321-442); C, Pex5p(457-639).
39
Figure Legends:
Figure 1: Sequence/structure relationships in (A) human Pex5p(C) and (B) human
mSCP2. The positions of labeled secondary structural elements are depicted by
cylinders and arrows. Color coding, Pex5p(C), panel A: TPR1-TPR3, cyan; TPR4,
green; TPR5-TPR7, blue; 7C-loop, connecting TPR7 and the C-terminal helical bundle,
red; C-terminus, maroon. Color coding, mSCP2, panel B: Core domain, yellow; C-
terminus including PTS1 motif, orange. Residues of Pex5p and SCP2 involved in cargo
and receptor binding, respectively, have been identified using the program AREAMOL
of the CCP4 suite (Collaborative Computational Project, Number 4, (1994) and are
indicated in colors matching the bound sequence segments. Conserved residues have
been identified from multiple sequence alignments using BLAST/MVIEW (Brown et
al., 1998). In the ‘cons’ line, residues exhibiting 90% and 70 % homology to the
available sequences are indicated by upper case and lower case characters, respectively.
Residues that were identified by (Klein et al., 2001) and (Shimozawa et al., 1999) as
being involved in Pex5p receptor-cargo interactions are shown in red and blue colors.
TPR motif signature residues according to the criteria of (D'Andrea and Regan, 2003)
are underlined. Residue segments that function as hinge regions (496-500, 523-524,
533-537), triggering the conformational changes observed for the cargo-loaded and apo-
Pex5p(C) receptor are highlighted by orange bars that have been inserted into the
corresponding secondary structural elements.
Figure 2: Lipid binding to mSCP2 in the absence and presence of Pex5p(C), (A, B).
Binding of a spin labeled lipid molecule (5-doxylstearic acid, 5DSA) attenuates the
peak intensity in 1H,15N correlation spectra due to paramagnetic relaxation enhancement
40
(PRE). Spectra in the presence of oxidized (i.e. paramagnetic) and reduced lipid are
shown in green and black, respectively. Residues Thr105 and Gly106, which are located
in the centre of the lipid binding pocket, are entirely bleached. Gly139, which is
proximal to the PTS1 motif, experiences a large chemical shift perturbation in the
presence of Pex5p(C). It is less bleached in the Pex5p(C) complex, consistent with the
strongly reduced mobility of the C-terminal residues and the increased distance to the
lipid ligand. (C, D) Comparison of the lipid binding pocket of mSCP2, in the presence /
absence of Pex5p(C). The degree of attenuation of NMR signals due to PRE is colored
in green on a ribbon representation of mSCP2. Amide protons of residues with a more
than seven-fold reduction in peak intensities are depicted by green spheres.
Figure 3: Structures of the peroxisomal import receptor Pex5p(C) in the presence
(A,B,D) and in the absence (C,D) of the cargo mSCP2. Color coding, Pex5p(C):
TPR1-TPR3, cyan; TPR4, green; TPR5-TPR7, blue; 7C-loop, connecting TPR7 and the
C-terminal helical bundle, red; C-terminus, maroon. Color coding, mSCP2: Core
domain, yellow; C-terminus including PTS1 motif, orange. The orientation of the
receptor in (A) and (C) is identical. The ribbon of the Pex5p(C)/mSCP2 complex in (B)
has been rotated by 600 around a horizontal axis within the paper plane with respect to
the orientation in (A), to illustrate the mode of mSCP2 binding to the receptor. (D)
Superimposed Pex5p(C) receptor structures in the presence and in the absence of
mSCP2. The colors of the trace of the cargo-loaded conformation are as in panels A-C,
except that the conformational hinge regions are colored in orange. The trace of the apo-
Pex5p(C) structure is in gray, except for the 7C-loop, which is colored in faint red. The
coordinates of TPR segments 1-4 were used for structural superposition using the
program SSM (Krissinel and Henrick, 2004) (rmsd = 0.78 Å for 164 common residues).
41
The largest structural deviations of up to 8 Å are observed at the 7C-loop and adjacent
regions and are indicated by a red arrow.
Figure 4: Surface presentations of the peroxisomal import receptor Pex5p(C), in
the presence (A-B) and in the absence (C) of the cargo mSCP2. The right panel
structures are rotated by 45° with respect to those in the left panels by a horizontal axis
within the paper plane. The color codes are as in Figure 3. While the structure of the
Pex5p(C)/mSCP2 complex is shown in (A), only the structure of the cargo-loaded
conformation of the Pex5p(C) receptor is displayed in (B). The PTS1 and secondary
mSCP2 binding areas are mapped onto the Pex5p(C) surface in their respective colors
(orange, yellow). In the structure of the apo-Pex5p(C) receptor, the approximate
location of the PTS1 binding site, as determined from the Pex5p(C)/mSCP2 complex, is
indicated by an orange circle. Conformational changes of several residues at this site
lead to disappearance of the open tunnel, observed in the Pex5p(C)/mSCP2 complex
(B). In the apo-conformation, the 7C-loop region (red) is well separated from the
remaining TPR segments of the receptor.
Figure 5: Structural determinants of mSCP2 cargo loading onto Pex5p(C). (A)
Stereo view of the 2FO-FC electron density, using phases from the refined model and
contoured at 1 of the PTS1 motif from mSCP2 (gray) and some interacting residues
from Pex5p and ordered solvent molecules (dark green). (B) Pex5p(C)/mSCP2 complex
formation by two distinct interfaces; C-terminal PTS1 motif from mSCP2 (orange)–
central cavity of the circular TPR motif structure from Pex5p; secondary surface from
mSCP2–C-terminal helical bundle from Pex5p. Ser600 is in a central position between
the two surface patches, allowing the proper arrangement of the two cargo surface
patches of Pex5p to support binding of mSCP2. The C-terminus of the 7C-loop (red)
42
interacts by a few hydrogen bonds with the TPR1 segment. (C) TPR4 motif of
Pex5p(C), as observed in the cargo-loaded structure of the receptor. Specific
interactions between TPR3 and TPR4, generating a circular conformation of Pex5p(C),
are shown. Colors are as in Figures 3 and 4, except that some of the bonds of residues
from the C-terminal TPR motifs 5-7 and the 7C-loop are colored in gray to allow
illustrations of oxygen and nitrogen atoms. Hydrogen bonds are shown by dashed lines.
Figure 6: 7C- loop mutants lead to functional PTS1 import defects
Pex5p-deficient fibroblast cells from Zellweger patient PBD005 were co-transfected
with a PTS2-tagged CAcT expressing plasmid, pEGFP-SCP2 and plasmids expressing
either wt Pex5p or a range of different single residue mutants (N382A, Q586R, S589Y,
S600W). At 24 hours after transfection PTS2-CAcT (A, red color) and endogenous
catalase (B, red color) were labeled by immunofluorescence while EGFP-SCP2 was
detected by direct fluorescence (A and B, green color). In cells expressing wt Pex5p,
both marker proteins and EGFP-SCP2, co-localized in peroxisomes (A and B, yellow
color). All Pex5p mutants were capable to restore the PTS2 import defect of PEX5
deficient cells. In contrast, all mutants were impaired in catalase import and showed
more or less pronounced import defects for EGFP-SCP2. The strongest effect was
observed for the S600W mutant that led to complete mislocalization of both PTS1
proteins into the cytosol. The introduction of the 7C-loop mutation Q586R resulted in
an inefficient SCP2 import as indicated by the diffuse cytosolic staining and the very
few peroxisomes labeled in the representative cells. Strikingly, in cells expressing the
Pex5p mutants S589Y and N382A, both cytosolic and peroxisomal localizations of
SCP2 were found while the same cells were devoid of functional catalase import as
indicated by the lack of a congruent punctuate pattern.
α1Α α1Β α2Α α2Β
320 330 340 350 360 370 380 390 400 | | | | | | | H H | |Pex5 LTSATYDKGYQFEEENPLRDHPQPFEEGLRRLQEGDLPNAVLLFEAAVQQDPKHMEAWQYLGTTQAENEQELLAISALRRCLELKPDNQTcons ---------------np----------G------G-l--a-l--Eaa----P---eaW--LG------ene--ai-A------l-p-n--
α3Α α3Β α4Α α4B α5Α
410 420 430 440 450 460 470 480 490 |H H | | | | | | | |Pex5 ALMALAVSFTNESLQRQACETLRDWLRYTPAYAHLVTPAEEGAGGAGLGPSKRILGSLLSDSLFLEVKELFLAAVRLDPTSIDPDVQCGLcons al--La-s-tn------A---L--w----p-y----------------------------------v--l---A----p---D-D----L
α5Α α5Β α6Α α6Β α7Α α7Β
500 510 520 530 540 550 560 570 580 H H| | | H H H | H | H H | . H. H H |Pex5 GVLFNLSGEYDKAVDCFTAALSVRPNDYLLWNKLGATLANGNQSEEAVAAYRRALELQPGYIRSRYNLGISCINLGAHREAVEHFLEALNcons GvL--L--e---a---f--Al---P-d---Wn-lGa-lAN---s-eA--AY--AL---P---r--yN-g-s--n-g---ea------al-
αC1 αC2 αC3
590 600 610 620 630 | | . .H H | H H H H H |Pex5 MQRKSRGPRGEGGAMSENIWSTLRLALSMLGQSDAYGAADARDLSTLLTMFGLPQcons -------------------w--L--------------------------------
α1 α2 β1 β2 β3
10 20 30 40 50 60 70 80 90 | | H | H H H | | | | | |SCP2 MGFPEAASSFRTHQIEAVPTSSASDGFKANLVFKEIEKKLEEEGEQFVKKIGGIFAFKVKDGPGGKEATWVVDVKNGKGSVLPNSDKKADcons -----------------------------------------------vkk-------------------W--D-K-g-g---------aD
β4 α3 α4 β5 α5
100 110 120 130 140 | H H | | | LQ | SCP2 CTITMADSDFLALMTGKMNPQSAFFQGKLKITGNMGLAMKLQNLQLQPGNAKL Figure 1cons ------d-df----------------GK-K--Gn--l--KL------------ Stanley et al.
Figure 1
Figure 2
1 2 3 4 5 6 7 C
A B
C D
Figure 3 Stanley et al.
Figure 3
45 deg.
A: Pex5p (cargo) -mSCP2
B: Pex5p (cargo)
C: Pex5p (apo)
Figure 4 Stanley et al.
Figure 4
Figure 5
Figure 6
1
Recognition of a functional peroxisome type 1 target
by the dynamic import receptor Pex5p
Will A. Stanley1, Fabian V. Filipp2, Petri Kursula1, Nicole Schüller1, Ralf
Erdmann3, Wolfgang Schliebs3, Michael Sattler2, Matthias Wilmanns1*
1 EMBL-Hamburg Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany
2 Structural and Computational Biology Unit, EMBL-Heidelberg, Meyerhofstrasse 1,
69117 Heidelberg, Germany
3 Institute for Physiological Chemistry, Department of Systems Biology, Faculty of
Medicine, Ruhr University of Bochum, 44780 Bochum, Germany.
Supplementary Material
F) Supplemental Text and Figures
2
Supplementary Figure S1: NMR data characterizing SCP2-Pex5p(C) binding.
(A, B) Chemical shift difference (Δδ) vs. residue number between the free and
Pex5p(C) bound state of 0.5 mM 15N, 2H-labeled preSCP2 (A) and mSCP2 (B) were
monitored at a 1:1.2 cargo/receptor ratio. The PTS1 and secondary interactions sites
are depicted by red and blue bars, respectively. Residues with Δδ > 0.1 ppm are
colored red or blue in Figure 2D. Secondary structure elements of SCP2 are shown on
top. (C) Comparison of 15N T1ρ relaxation times for free mSCP2 and when bound to
Pex5p(C) measured at 22˚C at 600 MHz 15N frequency. The T1ρ values of flexible
terminal regions are significantly higher than the average values of residues in the
core of the domain. Due to the interaction with Pex5p(C), T1ρ values of residues in
the PTS1 tail are strongly reduced, indicating that they become highly ordered. The
large increase in the molecular weight of mSCP2 bound to Pex5p(C) (49.6 kDa)
versus free mSCP2 (13.4 kDa) results in slower molecular tumbling, which is
reflected in a general reduction of the average T1ρ values. (D) Strong chemical shift
perturbations of N-H NMR signals (Δδ >0.1 ppm) are colored on a surface
representation of mSCP2. The PTS1 interaction site is shown in red; the secondary
binding surface is shown in blue.
Supplementary Figure S2: (A, B) Relative peak intensities in 1H,15N TROSY
experiments recorded on free preSCP2 (A) and when bound to Pex5p(C) (B),
indicating that the pre-sequence remains highly flexible even when SCP2 is bound to
Pex5p(C). (C) {1H}-15N heteronuclear NOE data for free preSCP2 at 295 K as
described (Farrow et al. 2003). Chemical shift assignments for residues 1-25 were
obtained from triple resonance experiments on free preSCP2 and 15N-edited NOESY
3
experiments on free and Pex5p(C) bound preSCP2. Spectra were processed with
NMRPipe and analysed using NMRVIEW.
Supplementary Figure S3: Structural mobility changes in the Pex5p receptor
and mSCP2 upon receptor/cargo complex formation. (A), sequence/B-factor plot
of the structures of Pex5p(C) in the absence (gray, black) and in the presence of the
mSCP2 cargo (red). The residue B factors of the two Pex5p(C) molecules with well
defined N-terminal TPR segments 1-4 are shown by filled symbols. The residue B
factors of the two other Pex5p(C) molecules, in which the N-terminal TPR domains 1-
4 are mobile, are displayed with open symbols. (B), sequence/B-factor plot of the
structures of mSCP2 in the absence (black) and in the presence (red) of the Pex5p(C)
receptor. The residue B factors of the cargo-unloaded mSCP2 structure have been
taken from the coordinates of the PDB entry 1C44 (Choinowski et al., 2000). In (A)
and (B), the B factors of the Cα positions have been used for display. (C), ribbon
representations of the conformations of mSCP2 loaded onto the Pex5p(C) receptor
(left) and unloaded (right). The residue B factors are mapped on the two ribbons in
rainbow colors, ranging from blue (B = 15 Å2) to red (B = 60 Å2). While the N-
termini of mSCP2 are flexible in both conformations, the C-terminus bearing the
PTS1 motifs becomes the most rigid part of the structure upon loading onto the
Pex5p(C) receptor. The C-terminus of mSCP2 straightens into an extended
conformation pointing away from the remaining structure.
Supplementary Figure S4: Expression of Pex5p variants in human fibroblasts. Equal
amounts of whole cell lysates (10 ug protein) from Pex5p-free human fibroblasts
transfected transiently for 24 hours with plasmids expressing (1) wt Pex5p, (2)
4
S600W, (3) Q586R, (4) S589Y, (5) N382A, (6) empty pcDNA vector and from wild-
type cells (7) were subjected to Western-Blot analysis using antibodies directed
against human Pex5p and GFP. Monoclonal anti-GFP (JL-8) antibodies were obtained
from BD Biosciences, Pharmingen, Germany.
1 2 3 4 5 6 7 C
0
20
40
60
80
21 41 61 81 101 121 141
SCP2(Pex5p) SCP2(apo)
A
B C
Figure S3Stanley et al.
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1 2 3 4 5 6 7
Pex5p
GFP-SCP2
Figure S4 Stanley et al.