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.
*
Correspondence:
Email:
wilmanns@embl-hamburg.de
Phone:
+49-40-89902-110
Fax:
+49-40-89902-149
Running Title: Target recognition by the import receptor Pex5p
1
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 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.
2
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 so 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
3
import receptor peroxin Pex5p. Human diseases leading to impaired fatty acid
metabolism, organ dysfunction, and neonatal mortality, such as Zellweger’s syndrome,
are often caused by mutations in the Pex5p receptor (Weller et al., 2003), rendering the
receptor an important subject 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., 2003; 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 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 cargo 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
4
loose structural arrangement of the receptor tetratricopeptide repeat (TPR) segments and
by the intrinsic structural flexibility within some of these structural segments.
5
Results
Selection 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 SCP2, which contains a canonical Cterminal PTS1 motif (Seedorf et al., 1998). Its structure has been characterized
previously 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 in vivo and in vitro, allowing structural investigation of the receptor/cargo
complex.
We used NMR spectroscopy and isothermal titration microcalorimetry (ITC) to
determine the molecular basis of SCP2 receptor binding. For both 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 shifts and line widths indicate that the presequence remains
unstructured and dynamic and is not involved in receptor binding. The binding affinities
of preSCP2 and mSCP2 for the receptor, as measured by ITC, are both in the order of
100 nM (Table 1), indicating that the presequence is tolerated and does not affect the
receptor interaction.
6
To determine whether SCP2 retains its function upon loading onto the Pex5p import
receptor, we used two complementary approaches. First, we investigated the capacity of
SCP2 to bind specific lipids 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
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 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 sequence signature, is in a distorted arrangement
(Figure 5A) and is preceded by a glycine-rich loop that is, in part, flexible. The Cterminus 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
7
structure. The long loop connecting the seventh TPR segment and the C-terminus (589601), 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 cargoloaded 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 ringlike 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
region, the preceding residues (134-140) interact with the receptor by van der Waals
forces only.
8
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 a PTS1 peptide 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
Pex5p(C) (Supplement, Figure 1C). Significant chemical shift changes have also been
detected for residues in the secondary binding site.
9
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). Comparative analysis of the cargo-loaded and unloaded
Pex5p(C) receptor structures 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 no longer completes the 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,
10
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). 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
11
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 Cterminal PTS1 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
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 Nterminal 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).
To examine 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.
12
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 tenfold 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.
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, possibly with additional
requirements for import (Purdue and Lazarow, 1996). As a control, we tested Pex5pdependent
import
of
the
PTS2-tagged
reporter
protein,
chloramphenicol
acetyltransferase (CAcT) as well. To control for expression level and turnover of the
mutants, we have analyzed all Pex5p mutants in a Pex5p-free cell line 24 hours after
transfection with the appropriate plasmids by Western blotting (Supplement, Figure
S4), demonstrating that the Pex5p mutants were synthesized at their full-length and at
steady-state levels 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.
13
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, three of the 7C-loop mutants (Q586R, S589Y, S600W) 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 and S600W mutants. During the first 24 hours
after transfection the bulk of GFP-SCP2 remained in the cytosol and only a few
peroxisomes were detected by characteristic punctate fluorescence (Figure 6). The
number of SCP2 containing peroxisomes further increased with incubation time and
after two to four days nearly all peroxisomes were labeled (data not shown).
By taking the our in vitro and in vivo data together, both lines of evidence demonstrate
critical contributions of several residues from the 7C-loop in the peroxisomal import of
PTS1 targets by the Pex5p receptor. Comparison of the data for SCP2 and catalase
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).
Discussion
Pex5p receptor recognition of diverse PTS1 targets
14
To what extent may our structural findings of the Pex5p receptor suggest general
principles 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 Cterminal 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 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 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 capacity to translocate 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
15
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 (by oligomerization, for
example) 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 proteins consists
of four steps per 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/release events that may be associated with 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, loading/unloading of the
cargo seems to be confined to the C-terminal part of the receptor. Data on the Nterminal part of Pex5p, which is thought to be largely unstructured (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., 2003; Schäfer et al., 2004). The model implies that these
interactions could have an effect on the affinity of the peroxisomal target for the C16
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.
Conclusions and Future Perspectives
The data presented here have led to three key findings with functional implications of
the molecular recognition process of PTS1 cargos by the Pex5p import receptor. (A)
The C-terminal part of the receptor undergoes substantial conformational changes upon
cargo binding. As demonstrated by structural comparison and supported by the in vitro
and in vivo analysis of several single residue mutations the loop C-terminal to the TPR
segment region, referred as to 7C-loop, plays a central role in altering the conformation
of the receptor upon cargo binding. (B) Within the cargo used in this investigation,
SCP2, there is a conformational change upon receptor binding, leading to disassembly
of the C-terminal PTS1 motif from the surface of the globular domain of the cargo.
Although this type of changes needs to be confirmed in different receptor/cargo
complexes, our data suggest that similar “unwinding” may occur at the C-termini of
other PTS1 proteins. (C) The structure of the Pex5p(C)/SCP2 complex demonstrates
that the receptor/ligand interactions for PTS1-containing cargos is not restricted to this
C-terminal recognition motif. A future challenge remains in the determination of the
events leading to cargo release as a subsequent functional step in the translocation cycle
17
of Pex5p. Further, it will be of specific interest to investigate the functional implications
of potential release factors such as Pex8p and changes of the receptor environment, by
association/dissociation of the receptor with the peroxisomal membrane during the
translocation cycle.
18
Experimental Procedures
Protein preparation
Human Pex5p(C) (residues 315-639), preSCP2 (1-143), and mSCP2 (21-143) were
expressed from a modified pET24d vector (G. Stier, EMBL-Heidelberg) in E. coli
BL21(DE3). Mutants N382A, Q586R, S589Y and S600W were introduced into
Pex5p(C) using the QuickChangeXL 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. Cleared lysates were loaded onto a glutathione Sepharose 4B resin
(GE Healthcare) and eluted with 20 mM reduced glutathione. Fusion proteins were
cleaved with His6-TEV and applied to Ni-NTA agarose (QIAgen). The flow-through
was subjected to gel filtration through a Superdex 75 (16/60) column (GE Healthcare).
Crystallization and X-ray structure determination
Pex5p(C) and mSCP2 were mixed in a 2:3 molar ratio and dialyzed against 20 mM bisTris-propane, 20 mM KCl, and 1 mM TCEP (pH 7.0). The protein mixture was
concentrated to 7 mg ml-1 by ultrafiltration. Crystallization was carried out by mixing 1
l protein with 1 l reservoir solution (24% (w/v) PEG 3350, 175 mM NaCl and 100
mM bis-Tris, pH 6.5), using hanging drop vapor diffusion at 20 °C. Prior to X-ray data
collection, 10 % (v/v) PEG 400 was added to the drop for 5-10 min. Crystals of
unliganded Pex5p(C) were obtained by mixing 1 l protein with 1 l reservoir solution
19
(23% (w/v) PEG 3350, 100 mM Tris-HCl (pH 8.75), and 0.22 mM octaethylene glycol
monolauryl ether) using hanging drop vapor diffusion at 20 °C.
X-ray data were collected at 100K at the synchrotron beamlines X13 (EMBL/DESY,
Hamburg, Germany), and at BL14.1 (BESSY, Berlin, Germany). Data were processed
and scaled using XDS (Kabsch, 1988). 5% of the reflections were randomly selected for
cross-validation. The structure of Pex5p(C)/mSCP2 complex was solved by molecular
replacement using MOLREP (Vagin and Teplyakov, 1997). Pex5p(C) and SCP2 were
located using PDB entries 1FCH and 1C44, respectively, as models. REFMAC
(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 added both manually and by ARP/wARP (Lamzin and
Wilson, 1993). The structure quality was assessed using PROCHECK (Laskowski et al.,
1993). Residues 335-440 and 454-639 of Pex5p and residues 22-143 of SCP2 were
included in the final model.
In order to determine the unliganded Pex5p(C) structure, the Pex5p coordinates from
the complex structure were split into two parts, spanning residues 335-440 and 454-637,
to find a molecular replacement solution using MOLREP (1994; Vagin and Teplyakov,
1997). All four copies of the C-terminal part, but only two copies of the N-terminal part
could be located. After initial refinement (Murshudov, 1997) and rebuilding (Jones et
al., 1991), weak electron density was observed for the two missing N-terminal
fragments, allowing the determination of the overall orientation of each domain. The
orientation of each N-terminal part relative to the C-terminal part was essentially the
same in all four Pex5p molecules. Refinement was continued by applying NCS
20
restraints separately to the N- and C-terminal halves of each Pex5p monomer.
Furthermore, TLS parameters were applied during refinement. Due to the flexibility of
the N-terminal parts of two of the four Pex5p molecules in the crystal and the
anisotropy of the 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
Isotopically-labeled (90% 2H, 13C and/or
15
N) SCP2 was prepared by growing bacteria
in minimal medium supplemented with [U-13C] glucose and/or
15
NH4Cl 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 methods (Sattler M et al., 1999). The assignments for
SCP2 in the 50 kDa SCP2/Pex5p(C) complexes were obtained using triple resonance
and
2
15
N-edited TROSY-NOESY experiments on samples comprising
2
H,15N- or
H,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
1
H,15N-TROSY experiments. 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).
21
Isothermal titration microcalorimetry
Proteins were co-dialyzed against 100 mM potassium phosphate (pH 7.4), 1 mM DTT.
When appropriate 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 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 expression vector pGD106 (Braverman
et al., 1998) by using the Quickchange XL – Site Directed Mutagenesis Kit
(Stratagene). GFP-SCP2 expression vector was derived from subcloning a PCR
amplification product of mSCP2 into pEGFP-C1 plasmid (Clontech Laboratories, Inc.).
All primers used are listed in Supplementary Table S1. pPTS2-CAcT encoding an Nterminal 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. Pex5p-deficient cells (Dodt et al., 1995) grown
22
for one day on cover-slides in 60 mm tissue-culture dishes were transfected with
pPTS2-CAcT, pEGFP-SCP2 and one of the Pex5p expression plasmids using
FuGENE 6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany). At
various time points (24, 48, 72, 96 hours after transfection) cells were fixed 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
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 a 900 MHz NMR, and BESSY,
Berlin, Germany, for access to the synchrotron radiation beamline BL14.1.
Accession Numbers
23
Coordinates and structure factors have been deposited at the Protein Data
Bank with accession codes 2C0L and 2C0M .
24
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31
Tables
Table 1: Thermodynamic characterization of Pex5p interaction with PTS1containing ligands by ITC.
Pex5p receptor
Cargo
H
TS
G
Kd
(kJ/mol)
(kJ/mol)
(kJ/mol)
(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.
32
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)
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
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
Pex5p(C) N/Cb
18/30
14, 23,76,76/15,19,41,42
SCP2
39
-
Solvent
24
15
Refinement statistics
Average B factors [Å2]a
Rmsd B factors of protein bonded atoms [Å2]
33
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
0.1
0.3
Table 2 Legend:
a
TLS refinement parameters have been applied.
b
N, Pex5p(321-442); C, Pex5p(457-639).
34
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; Cterminus 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 apoPex5p(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
35
(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 apoPex5p(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).
36
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)
37
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 introduction of the 7Cloop mutations Q586R and S600W resulted in an inefficient SCP2 import as indicated
by the diffuse cytosolic staining and only weak labeling of peroxisomes 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.
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