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

3Institute 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.

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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

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segment of the 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

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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 C-

terminal 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.

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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 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

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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

region, the preceding residues (134-140) interact with the receptor by van der Waals

forces only.

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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 (K d =

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.

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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,

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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

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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 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 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).

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.

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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.

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 Pex5p-

dependent 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.

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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

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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 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 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

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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 N-

terminal 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 C-

terminal part of the receptor, ultimately leading to cargo release. Pex8p appears to play 16

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

of Pex5p. Further, it will be of specific interest to investigate the functional implications

17

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.

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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 bis-

Tris-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 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 methods (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. 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 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. 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 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.

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)

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/Cb18/30 14, 23,76,76/15,19,41,42

SCP2 39 -

Solvent 24 15

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 0.3 0.1

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; 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 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 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).

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 7C-

loop 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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