Molecular Cell 24, 653–663, December 8, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.10.024

Recognition of a Functional PeroxisomeType 1 Target by the Dynamic Import Receptor Pex5p

Will A. Stanley,1 Fabian V. Filipp,2 Petri Kursula,1

Nicole Schuller,1 Ralf Erdmann,3 Wolfgang Schliebs,3

Michael Sattler,2 and Matthias Wilmanns1,*1European Molecular Biology Laboratory-Hamburg

OutstationNotkestrasse 8522603 Hamburg2Structural and Computational Biology UnitEuropean Molecular Biology Laboratory-HeidelbergMeyerhofstrasse 169117 Heidelberg3Department of Systems BiologyInstitute for Physiological ChemistryFaculty of MedicineRuhr University of Bochum44780 BochumGermany

Summary

Peroxisomes require the translocation of folded andfunctional target proteins of various sizes across the

peroxisomal membrane. We have investigated thestructure and function of the principal import receptor

Pex5p, which recognizes targets bearing a C-terminalperoxisomal targeting signal type 1. Crystal structures

of the receptor in the presence and absence of a perox-isomal target, sterol carrier protein 2, reveal major

structural changes from an open, snail-like confor-mation into a closed, circular conformation. These

changes are caused by a long loop C terminal to the7-fold tetratricopeptide repeat segments. Mutations

in residues of this loop lead to defects in peroxisomalimport in human fibroblasts. The structure of the re-

ceptor/cargo complex demonstrates that the primaryreceptor-binding site of the cargo is structurally and

topologically autonomous, enabling the cargo to re-tain its native structure and function.

Introduction

Diverse machineries involved in translocating proteinsacross organellar membranes are required to maintainthe compartmentalization of biological processes withineukaryotic cells (Kunau et al., 2001; Wickner and Schek-man, 2005). Many components of membrane receptorsrecognize specific targeting sequences in proteins des-tined for translocation (Eichler and Irihimovitch, 2003).However, they differ in their requirements for cargo fold-ing/unfolding during the translocation process to retainthe functional integrity of the cargo. To date, the onlywell-characterized system supporting the translocationof folded protein targets is the nuclear import/exportsystem by karyopherins (Conti and Izaurralde, 2001;

*Correspondence: wilmanns@embl-hamburg.de

Matsuura and Stewart, 2004). The extent to which othertranslocation systems may resemble karyopherin-medi-ated processes remains elusive.

Peroxisomal import is one of the few transport pro-cesses that uses a translocon for the purpose of traf-ficking folded and functional cargo proteins acrossmembranes (Gould and Collins, 2002; Holroyd andErdmann, 2001; Lazarow, 2003; Schnell, 2000; van derKlei and Veenhuis, 2002). To date, more than two dozenproteins involved in trafficking cargo to the peroxi-some—referred to as ‘‘peroxins’’—have been identifiedand partially characterized. No pore-like structure inthe peroxisomal membrane has so far been observed,and the exact composition of the import translocon,possibly assembled according to the size and type ofimport substrates, has yet to be established. The major-ity of peroxisomal matrix proteins destined for trans-location into peroxisomes share the C-terminal type 1peroxisomal targeting signal (PTS1) motif. It comprisesan obligatory C-terminal tripeptide, conforming to theconsensus sequence -[S/A/C]-[K/H/R]-[L/M]-CO2

2, whichis specifically recognized by the C-terminal segment ofthe import receptor peroxin Pex5p. Human diseasesleading to impaired fatty acid metabolism, organ dys-function, and neonatal mortality, such as Zellweger’ssyndrome, are often caused by mutations in the Pex5preceptor (Weller et al., 2003), rendering the receptor animportant subject for biomedical research.

At the molecular level, these Pex5p translocation tar-gets appear to be released into the peroxisomal lumenby interactions between the receptor and other peroxinsand by their association with the peroxisomal membrane(Gouveia et al., 2003; Holroyd and Erdmann, 2001;Madrid et al., 2004). Cargo loading may also influencethe oligomeric state of Pex5p and its interactions withother peroxisomal membrane docking factors, such asPex14p (Madrid et al., 2004; Wang et al., 2003). Otherevidence demonstrates that the cargo-loaded Pex5preceptor may even shuttle across the peroxisomal mem-brane (Dammai and Subramani, 2001). However, to date,questions still remain regarding whether the receptor, orparts of the receptor, physically shuttle or just becomeaccessible to the peroxisomal lumen (Erdmann andSchliebs, 2005; Kunau et al., 2001).

To investigate the molecular requirements of thisdynamic receptor for both cargo loading and release,we determined the structures of the PTS1-cargo bindingregion of the Pex5p receptor in the presence and in theabsence of a peroxisome translocation target, sterol car-rier protein 2 (SCP2). The cargo is bound to the receptorby two separate binding sites—a C-terminal PTS1 motifand a topologically separate secondary site—providinga rationale as to how the target remains folded and func-tional during translocation. A comparative analysis of thetwo Pex5p receptor structures reveals major conforma-tional changes in the receptor upon cargo loading, whichare generated by the loose structural arrangement of thereceptor tetratricopeptide repeat (TPR) segments andby the intrinsic structural flexibility within some of thesestructural segments.

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Results

Selection of a Functional Receptor/Cargo SystemIn order to unravel the molecular basis of PTS1-drivenprotein translocation via the Pex5p receptor into perox-isomes, we searched for a suitable physiological targetthat could serve as a model system. We selectedSCP2, which contains a canonical C-terminal PTS1 motif(Seedorf et al., 1998). Its structure has been character-ized previously by X-ray crystallography and NMR spec-troscopy (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-terminalthiolase domain and a C-terminal SCP2 domain, andpreSCP2, a protein with a molecular mass of about 15kDa, which is processed into its mature form (mSCP2)by proteolytic cleavage of a 20 residue leader sequenceafter translocation into peroxisomes (Figure 1). SCP2binds to the Pex5p receptor both in vivo and in vitro,allowing structural investigation of the receptor/cargocomplex.

We used NMR spectroscopy and isothermal titrationmicrocalorimetry (ITC) to determine the molecular basisof SCP2 receptor binding. For both preSCP2 andmSCP2, respectively, the chemical shift perturbationsin 1H,15N correlation spectra upon binding to Pex5p(C)affect the same set of SCP2 residues (see Figures S1and S2 in the Supplemental Data available with this arti-cle online). Furthermore, chemical shifts and line widthsindicate that the presequence remains unstructured anddynamic and is not involved in receptor binding. Thebinding affinities of preSCP2 and mSCP2 for the recep-tor, as measured by ITC, are both in the order of 100 nM(Table 1), indicating that the presequence is toleratedand does not affect the receptor interaction.

To determine whether SCP2 retains its function uponloading onto the Pex5p import receptor, we used twocomplementary approaches. First, we investigated thecapacity of SCP2 to bind specific lipids in the presenceand in the absence of Pex5p(C). To locate the lipid-bind-ing site with SCP2, we used spin-labeled paramagneticdoxylstearate as a fatty acid derivative (Garcia et al.,2000). We observed similar bleaching of NMR signalsupon binding of this fatty acid derivative to either freeSCP2 or Pex5p(C)-bound SCP2, indicating that the func-tional integrity of SCP2, in terms of lipid binding, is notimpaired upon receptor loading (Figure 2). We furtherused ITC to quantify the thermodynamic parametersgoverning the receptor/cargo interaction in the presenceand in the absence of stearoyl-CoA, a physiologicalligand of SCP2 (Frolov et al., 1996). The data show thatmSCP2 binds to the receptor with about the same Kd

and 1:1 stoichiometry, regardless of whether it is loadedwith stearoyl-CoA (Table 1).

Structure of the Cargo-Loaded Pex5p ReceptorWe have determined the crystal structure of the C-termi-nal part of the Pex5p import receptor (Pex5p(C), residues315–639) in the presence of mSCP2 (21–143), at 2.3 A res-olution (Figures 1, 3A, 3B, 4A, 4B, and 5; Table 2). TheX-ray data reveal the complete structure, except for thevery N terminus (315–334) and one loop (441–453) ofPex5p(C). Most of its structure is formed by seven con-secutive TPR motifs, each consisting of a helix-turn-helix

motif (D’Andrea and Regan, 2003). The fourth segment,which matches the established TPR sequence signature,is in a distorted arrangement (Figure 5A) and is precededby a glycine-rich loop that is, in part, flexible. The C termi-nus 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 struc-ture. The long loop connecting the seventh TPR segmentand 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 segmentlink the two arch-shaped TPR motif triplets (1–3 and5–7), thus generating a pseudocircular structure of thecargo-loaded receptor with a tunnel in its center, whichis open to both faces of the ring-like structure (Figure 4B).The connecting segments represent the most mobileregions of the cargo-loaded receptor (Figure S3A) and,therefore, can be regarded as hinges. The C-terminalhelical bundle does not participate in the circular ar-rangement (Figures 3A, 3B, 5A, and 5B).

The structure of the receptor-bound mSCP2 resem-bles that of free SCP2 (Choinowski et al., 2000), exceptthe C terminus that bears the PTS1 motif (Figure S3C).In the Pex5p(C) receptor complex, the ten C-terminalresidues (134–143) of mSCP2 adopt an extended confor-mation, pointing away from its core domain. However,unlike the structure of free SCP2, wherein both terminiare flexible, in the receptor-bound SCP2 structure, theC terminus becomes the most rigid part of the overallstructure, while its N terminus remains mobile (Figure 1Cand Figures S3B and S3C). The most C-terminal AKLmotif (141–143) of mSCP2 binds within the central holeof the ring-like structure of the receptor. It is involved inspecific interactions with four asparagines (N415, N526,N534, and N561) that are located on the N-terminalhelices of TPR segments 4, 5, 6, and 7. These residuesare conserved (Figures 1, 5A, and 5B), indicating thatPTS1 binding is a general property of the Pex5p receptor.In contrast to the most C-terminal region, the precedingresidues (134–140) interact with the receptor by van derWaals forces only.

The structure of the receptor/cargo complex also re-veals a second interaction site of about 500 A2 that isformed by the C-terminal helical bundle of Pex5p(C)and a surface patch of SCP2, covering parts of helicesa1 and a3 (Figures 1, 4A, 4B, and 5B). ITC binding datausing a PTS1 peptide reveal that its binding affinity toPex5p(C) is reduced (Kd = 664 nM) compared with the en-tire protein cargo (Table 1). Thus, the data indicate thatthere is a notable contribution by the secondary interfacein SCP2 loading onto the Pex5p receptor. However, noneof the residues of either the Pex5p receptor or SCP2involved in these interactions are invariant (Figure 1).These findings suggest that, in contrast to the PTS1-mediated receptor/cargo interactions, the formation ofthe secondary SCP2/Pex5p(C) interface is specific andmay not be conserved taxonomally. Since PTS1 targetsare generally unrelated in terms of structure and func-tion, with the exception of the C-terminal PTS1-receptorrecognition motif, our findings suggest that the involve-ment of secondary binding sites may serve as a deter-minant for the sorting of folded import substrates.

Our NMR spectroscopy data on mSCP2 in the pres-ence and in the absence of the receptor correlate with

Target Recognition by the Import Receptor Pex5p655

Figure 1. Sequence/Structure Relationships in Human Pex5p(C) and Human mSCP2

The positions of labeled secondary structural elements are depicted by cylinders and arrows. Color coding for Pex5p(C) in (A) is as follows:

TPR1-TPR3, cyan; TPR4, green; TPR5-TPR7, blue; 7C loop, connecting TPR7 and the C-terminal helical bundle, red; and C terminus, maroon.

Color coding for mSCP2 in (B) is as follows: core domain, yellow; and C terminus including PTS1 motif, orange. Residues of Pex5p and SCP2

involved in cargo and receptor binding, respectively, have been identified using the program AREAIMOL of the CCP4 suite (CCP4, 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 to 524, and 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.

the crystallographic analysis. The largest chemical shiftperturbations are found for the backbone amides ofthe C terminus (136–143) (Figure S1B), coinciding withthe structural alterations observed by comparing thestructures of unbound SCP2 and Pex5p(C)-boundSCP2 (Figure S3C). NMR relaxation measurements indi-cate that the PTS1 backbone, which is flexible and dis-ordered in free SCP2, rigidifies upon binding to Pex5p(C)(Figure S1C). Significant chemical shift changeshave also been detected for residues in the secondarybinding site.

Taken together, our biochemical and structural datasuggest bipartite binding of the SCP2 cargo to the Pex5preceptor. The PTS1-binding site at the most C-terminalregion is structurally and topologically well separatedfrom the functional SCP2 core domain. As such, the dataprovide a rationale for our observations on a mechanismof receptor recognition that does not interfere with thefunction of SCP2 as lipid-binding protein. Our data, how-ever, do not support previous hypotheses proposing thatbinding of lipid substrates to members of the SCP2 familymay enhance the exposure of the PTS1 motif, thereby

Table 1. Thermodynamic Characterization of Pex5p Interaction with PTS1-Containing Ligands by ITC

Pex5p Receptor Cargo DH (kJ/mol) TDS (kJ/mol) DG (kJ/mol) Kd (nM)

WT mSCP2 242.4 21.2 241.2 109 6 34

WT mSCP2(SCoA) 231.8 8.9 240.8 124 6 17

WT preSCP2 235.9 6.2 242.1 74 6 9

WT PGNAKL 245.1 28.7 236.4 664 6 37

N382A mSCP2 227.3 10.8 238.1 348 6 54

Q586R mSCP2 217.4 17.3 234.5 1343 6 321

S589Y mSCP2 238.7 1.20 239.9 173 6 23

S600W mSCP2 no binding

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.

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Figure 2. Lipid Binding to mSCP2 in the Absence and Presence of Pex5p(C)

(A and B) Binding of a spin-labeled lipid molecule (5-doxylstearic acid, 5DSA) attenuates the peak intensity in 1H,15N correlation spectra due to

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 center of the lipid-binding pocket, are entirely bleached. Gly139, which is proximal to the PTS1 motif, experi-

ences 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 and D) Comparison of the lipid-binding pocket of mSCP2 (Garcia et al., 2000), 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 7-fold reduction

in peak intensities are depicted by green spheres.

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 deli-cate balance between the binding and release of cargoproteins to and from the appropriate import receptor.To investigate the molecular parameters that governcargo release, we have also determined the structureof the import receptor Pex5p(C) in the absence of a cargoat 2.5 A resolution (Figures 3C and 4C; Table 2). Com-parative analysis of the cargo-loaded and unloadedPex5p(C) receptor structures reveals major conforma-tional changes upon cargo binding. Contrary to previoushypotheses proposing that the TPR4 segment is a flexi-ble hinge (Gatto et al., 2000), these changes originatefrom three clusters in TPR segments 5 and 6, renderinga rotation of about 20� of the C-terminal TPR segmentswith respect to N-terminal TPR segments (Figure 3D). Asa result of this conformational change, the 7C loop of theapo structure no longer completes the ring-like structureof Pex5p(C) as observed in the cargo complex, thus gen-erating an open, snail-like arrangement of the receptor

(Figures 3C and 4C). For instance, Gln586 and Ser600,which interact with residues from the TPR1 segment inthe cargo-loaded receptor (Figure 5B), have moved bymore than 8 A in the apo structure.

Since the overall arrangement of TPR segment-con-taining structures can be described as a superhelicalcoil or solenoid (D’Andrea and Regan, 2003; Jineket al., 2004), we compared the underlying structural pa-rameters of the cargo-free and cargo-loaded structuresof the Pex5p receptor. Our analysis revealed that thesuperhelical pitch is about 30 A in the apo structure,rather than 20 A in the Pex5p/SCP2 complex. Further-more, the N-terminal helices of TPR7 (556–568) and theC-terminal helical bundle (601–613), which bear severalresidues that are involved in binding of the cargo PTS1motif, are moved by the equivalent of about two a-helicalpitches with respect to the N-terminal TPR segments,leading to a displacement of part of the PTS1 motif bind-ing site by several angstroms (Figure 3D). These sugges-tions 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 ofentropy, during cargo binding (Table 1). Indeed, a recent

Target Recognition by the Import Receptor Pex5p657

study has demonstrated coupled folding and ligandbinding in at least one TPR array (Cliff et al., 2005).

Our model allows for speculation on further possiblestructural changes. Although TPR4 is not involved inthe conformational changes evident from our compara-tive analysis of the apo- and cargo-loaded Pex5p recep-tor structures, we cannot exclude the possibility thatthere are steps during the target-import cycle that affectthis receptor segment as well. For instance, completefolding of the distorted TPR4 motif into the canonicalTPR domain structure would only require minor changesin the flexible loop N terminal to the TPR4 segment(Figure 5A). The resulting overall structure could openup into a superhelical arrangement with a pitch in theorder of 55 A, reminiscent of previous observations inanother TPR segment-containing structure (Jineket al., 2004). In contrast to the observed changes in thereceptor structures in the presence and in the absenceof cargo, the overall conformation of the cargo-loadedPex5p(C) receptor structure remains virtually identicalregardless of whether it is bound only to the C-terminal

Figure 3. Structures of the Peroxisomal Import Receptor Pex5p(C)

in the Presence and in the Absence of the Cargo mSCP2

Color coding for Pex5p(C) is as follows: TPR1-TPR3, cyan; TPR4,

green; TPR5-TPR7, blue; 7C loop, connecting TPR7 and the C-termi-

nal helical bundle, red; and C terminus, maroon. Color coding for

mSCP2 is as follows: core domain, yellow; and 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 60� 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 struc-

tures in the presence and in the absence of mSCP2. The colors of the

trace of the cargo-loaded conformation are as in (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 A for 164 common residues). The

largest structural deviations of up to 8 A are observed at the 7C

loop and adjacent regions and are indicated by a red arrow.

PTS1 motif (Gatto et al., 2000) or to a complete cargotarget, as shown by the Pex5p/SCP2 complex.

Residues from the 7C Loop Are Critical for In VivoPTS1 Import

In contrast to several known peroxisome disease muta-tions wherein direct interactions with the PTS1 motifare abolished, a patient with an inherited peroxisomebiogenesis disorder, infantile Refsum disease, wasfound to be impaired in the import of proteins containingonly the AKL- and KANL-type PTS1 motifs, such as SCP2and catalase (Shimozawa et al., 1999). In this patient,dysfunctional import into peroxisomes is linked to muta-tion S600W in Pex5p. Comparative analysis of the cargo-loaded and unloaded structures of the Pex5p(C) recep-tor reveals that Ser600, which is situated at the base ofthe 7C loop, plays a central role in connecting the C-ter-minal and N-terminal TPR segments, to arrange thePTS1-binding site as well as the secondary binding siteat the C-terminal helical bundle in the Pex5p(C)/SCP2complex (Figure 5B).

To examine the involvement of the 7C loop in PTS1 tar-get import, we mutated three residues (Gln586, Ser589,and Ser600) that are involved in specific interactions ofthis loop with other parts of the receptor in the cargo-bound conformation (Figure 5B). As a control, we choseone single-residue mutant from the TPR2 segment(N382A), which previously was shown to be involved inPTS1 cargo import by the S. cerevisiae Pex5p receptor(Klein et al., 2001). First, we measured the binding affinity

Table 2. Crystallographic Statistics

Pex5p(C):mSCP2 Pex5p(C)

X-Ray Data Collection Statistics

Space group P212121 P1

Unit cell dimensions [A] 40.5, 68.6, 137.4 53.5, 85.6, 88.9,

71.2�, 90.0�, 73.4�

Resolution range [A] 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/s_(I) 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 [A] 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 [A] 0.006 0.013

Rmsd bond angles [�] 1.0 1.4

Average B factors [A2]a

Pex5p(C) N/Cb 18/30 14, 23, 76, 76/15,

19, 41, 42

SCP2 39 —

Solvent 24 15

Rmsd B factors of protein-bonded atoms [A2]

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

a TLS refinement parameters have been applied.b N, Pex5p (321–442); C, Pex5p (457–639).

Molecular Cell658

of the cargo SCP2 to the resulting Pex5p variants underin vitro conditions (Table 1). As expected, no bindingcould be detected for the S600W mutant, while a morethan ten-fold reduced binding affinity was observedfor the Q586R Pex5p variant, thus demonstrating thecritical involvement of this residue from the 7C loop inSCP2 cargo recognition as well. On the other hand, thecargo binding by the Pex5p S589Y was only slightlyreduced.

The same mutations were introduced in full-lengthPex5p, and the resulting variants were expressed ina fibroblast cell line devoid of endogenous PTS1 recep-tor. Transfected cells were analyzed by fluorescence mi-croscopy for their capacity to import two establishedPTS1 peroxisome targets, catalase and SCP2 (Shimo-zawa et al., 1999). While SCP2, due to its low molecularweight and absence of evidenced tendencies for oligo-merization, can be considered as a model target with

Figure 4. Surface Presentations of the Peroxisomal Import Recep-

tor Pex5p(C) in the Presence and in the Absence 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 con-

formation 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 (C), the approximate location of the

PTS1-binding site, as determined from the Pex5p(C)/mSCP2 com-

plex, 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.

only modest structural requirements for import in itsfunctional form, catalase forms a homotetrameric heme-containing assembly with a molecular mass of about 240kDa, possibly with additional requirements for import(Purdue and Lazarow, 1996). As a control, we testedPex5p-dependent import of the PTS2-tagged reporterprotein chloramphenicol acetyltransferase (CAcT) aswell. To control for expression level and turnover of themutants, we have analyzed all Pex5p mutants in aPex5p-free cell line 24 hr after transfection with theappropriate plasmids by western blotting (Figure S4),demonstrating that the Pex5p mutants were synthe-sized at their full length and at steady-state levels com-parable with WT Pex5p expressed from the sameplasmid. The levels of expression were significantly in-creased when compared with WT Pex5p under controlof its endogenous promotor.

The transfected WT Pex5p receptor correctly directedboth PTS1 targets, catalase and SCP2, as well as thePTS2-tagged CAcT into the peroxisomal matrix (Fig-ure 6). As expected, three of the 7C-loop mutants(Q586R, S589Y, and S600W) and the PTS1 referencemutant (N382A) mediated properly the import of PTS2proteins but showed severe defects for catalase target-ing. Retarded import was observed for SCP2, mostapparently when expressing the Q586R and S600Wmutants. During the first 24 hr after transfection, thebulk of GFP-SCP2 remained in the cytosol and onlya few peroxisomes were detected by characteristicpunctate fluorescence (Figure 6). The number of SCP2-containing peroxisomes further increased with incuba-tion time, and after 2–4 days nearly all peroxisomeswere labeled (data not shown).

By taking our in vitro and in vivo data together, bothlines of evidence demonstrate critical contributions ofseveral residues from the 7C loop in the peroxisomalimport of PTS1 targets by the Pex5p receptor. Compar-ison of the data for SCP2 and catalase indicates anamplification of the effect for the latter one, which isexpected to be more sensitive because of its oligomericarrangement and requirement for cofactor binding.Although a quantitative interpretation of this differentialeffect will only be possible once a structure of receptor-catalase cargo complex becomes available, it is intrigu-ing to hypothesize on additional sorting effects for theimport of different PTS1 cargoes, supporting or comple-menting previous observations (Kiel et al., 2005; Knottet al., 2000; Otera et al., 2002).

Discussion

Pex5p Receptor Recognition of Diverse PTS1Targets

To what extent may our structural findings of the Pex5preceptor suggest general principles for the translocationof a variety of folded proteins into peroxisomes? FormSCP2, our data suggest a bipartite recognition mecha-nism, via the C-terminal PTS1 motif and a secondary,less-conserved binding site that is distinct in terms ofsequence and structure. Much attention has previouslyfocused on residues preceding the C-terminal tripeptidePTS1 motif, suggesting that some of these may serve asdeterminants for altering binding affinity (Lametsch-wandtner et al., 1998; Neuberger et al., 2003). However,

Target Recognition by the Import Receptor Pex5p659

Figure 5. Structural Determinants of mSCP2

Cargo Loading onto Pex5p(C)

(A) Stereo view of the 2FO 2 FC electron den-

sity, using phases from the refined model and

contoured at 1s, 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; sec-

ondary surface from mSCP2-C-terminal heli-

cal 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) 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.

the structure of the Pex5p(C)/mSCP2 complex has notrevealed any further specific cargo interactions withthe receptor beyond the C-terminal PTS1 motif. Althoughseveral hydrophobic interactions can be observed,these interactions may change in other PTS1 targets, inwhich the orientation of the linker region between thePTS1 motif and the functional domain structures maybe different. As such, it is difficult to quantify possiblecontributions of residues from the linker connecting thecore domain of SCP2 and the PTS1 C terminus toPex5p receptor.

Recent data on the recognition of another peroxisometarget, alanine:glyoxylate aminotransferase (AGA), bythe Pex5p receptor support our findings on bipartitecargo binding by a second topologically remote inter-action site separated by about 40 residues in the AGAsequence (Huber et al., 2005). Since considerable varia-tion has been observed in the sequence within the PTS1motif of different targets, it is plausible to assume thatthere is a correlation between a weakened PTS1-Pex5preceptor interaction and a need for secondary bindingsites on the cargo surface. A peculiar property of thePex5p import system is its capacity to translocate largefolded proteins, either monomeric or oligomeric (Bro-card et al., 2003; Lazarow, 2003; Walton et al., 1995;Yang et al., 2001). Some PTS1 targets can be importedinto peroxisomes by either a PTS1-dependent or PTS1-

independent mechanism (Parkes et al., 2003). On theother hand, there has been recent biochemical andstructural evidence demonstrating that oligomerizationof proteins established as physiological peroxisomaltargets 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 olig-omerization, for example) appears to be more likely thanthe PTS1 motif, as predicted by previous data (Parkeset al., 2003) and supported by the Pex5p(C)/mSCP2complex structure reported here.

The Pex5p Import Receptor CycleThe generally accepted model for PTS1-driven importof peroxisomal proteins consists of four steps per cycle:(1) cargo recognition by the Pex5p receptor, (2) cargo-loaded receptor docking and, possibly, integration intothe peroxisomal membrane, (3) cargo release into theperoxisomal lumen, and (4) recycling of the unloaded re-ceptor into the cytosol (Erdmann and Schliebs, 2005).The model proposes two essential types of binding/re-lease events that may be associated with conforma-tional changes in the receptor: loading/unloading ofthe cargo and docking/release of the receptor to/fromthe peroxisomal membrane. For canonical PTS1-drivenimport, loading/unloading of the cargo seems to be con-fined to the C-terminal part of the receptor. Data on the

Molecular Cell660

Figure 6. 7C-Loop Mutants Lead to Functional PTS1 Import Defects

Pex5p-deficient fibroblast cells from Zellweger patient PBD005 were cotransfected 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, and S600W). At 24 hr

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

colocalized in peroxisomes ([A] and [B], yellow color). All Pex5p mutants were capable of restoring 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 intro-

duction 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 punctate pattern.

N-terminal part of Pex5p, which is thought to be largelyunstructured (Costa-Rodrigues et al., 2005), have indi-cated that this region is critically involved in membranedocking/release of the receptor, either by interactionswith other docking factors of the translocon, such asPex13p and Pex14p as well as Pex8p and Pex17p(only demonstrated in yeast), or by direct interactionswith the peroxisomal membrane (Agne et al., 2003; Gou-veia et al., 2003; Schafer et al., 2004). The model impliesthat these interactions could have an effect on the af-finity of the peroxisomal target for the C-terminal partof the receptor, ultimately leading to cargo release.Pex8p appears to play a key role in this process, eitheras a cargo release factor, as suggested from in vitrodata of the H. polymorpha Pex5p receptor (Wanget al., 2003) or as an inducer of a subsequent transloconcomplex with additional components allowing cargo re-lease (Agne et al., 2003). Regardless of the precise originof the conformational changes in the C-terminal part ofthe receptor, both the canonical nature of the loosearrangement of the TPR domains and our direct com-parative analysis of the cargo-loaded and unloaded

receptor structures suggest a ring-opening mechanismfor peroxisomal target release.

Conclusions and Future PerspectivesThe data presented here have led to three key findingswith functional implications for the molecular recognitionprocess of PTS1 cargoes by the Pex5p import receptor.(1) The C-terminal part of the receptor undergoes sub-stantial conformational changes upon cargo binding.As demonstrated by structural comparison and sup-ported by the in vitro and in vivo analysis of severalsingle-residue mutations, the loop C terminal to theTPR-segment region, referred to as the 7C loop, playsa central role in altering the conformation of the receptorupon cargo binding. (2) Within the cargo used in thisinvestigation, SCP2, there is a conformational changeupon receptor binding, leading to disassembly of theC-terminal PTS1 motif from the surface of the globulardomain of the cargo. Although this type of change needsto be confirmed in different receptor/cargo complexes,our data suggest that similar ‘‘unwinding’’ may occur atthe C termini of other PTS1 proteins. (3) The structure

Target Recognition by the Import Receptor Pex5p661

of the Pex5p(C)/SCP2 complex demonstrates that thereceptor/ligand interactions for PTS1-containing car-goes are not restricted to this C-terminal recognition mo-tif. A future challenge remains in the determination of theevents leading to cargo release as a subsequent func-tional step in the translocation cycle of Pex5p. Further,it will be of specific interest to investigate the functionalimplications of potential release factors such as Pex8pand changes of the receptor environment, by associa-tion/dissociation of the receptor with the peroxisomalmembrane during the translocation cycle.

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 Quik-

Change 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-buff-

ered LB medium supplemented with 1% (w/v) glucose, and induced

mid-log phase with 0.5 mM IPTG for 6 hr 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 flowthrough 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 ml21 by ultrafil-

tration. Crystallization was carried out by mixing 1 ml protein with 1 ml

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) (at 7 mg ml21) were

obtained by mixing 1 ml protein with 1 ml reservoir solution (23%

[w/v] PEG 3350, 100 mM Tris-HCl [pH 8.75], and 0.22 mM octa-

ethylene glycol monolauryl ether), using hanging drop vapor diffu-

sion at 20�C.

X-ray data were collected at 100 K at the synchrotron beamlines

X13 (EMBL/DESY, Hamburg, Germany), and at BL14.1 (BESSY, Ber-

lin, Germany). Data were processed and scaled using XDS (Kabsch,

1988). Five percent of the reflections were randomly selected for

crossvalidation. The structure of Pex5p(C)/mSCP2 complex was

solved by molecular replacement using MOLREP (Vagin and Teplya-

kov, 1997). Pex5p(C) and SCP2 were located using PDB entries

1FCH and 1C44, respectively, as models. REFMAC (Murshudov

et al., 1997) was used to refine the structure, applying TLS parame-

terization (Winn et al., 2001). Simulated annealing refinement was

carried out in CNS (Bruenger et al., 1998). Manual building and struc-

ture 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 (Mur-

shudov et al., 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 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 (CCP4, 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] glu-

cose 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 ac-

quired at 22�C (complex) or 37�C (free SCP2) on Bruker spectrome-

ters (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 et al., 1999). The assignments for SCP2 in

the 50 kDa Pex5p(C)/SCP2 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 (Dd = [(Dd1H)2 + (1/5

Dd15N)2]1/2, in ppm) were monitored in two-dimensional 1H,15N-

TROSY experiments. Spin-label-induced paramagnetic relaxation

enhancements (PREs) were analyzed from intensity changes in1H,15N-TROSY experiments of SCP2 or SCP2/Pex5p(C) recorded

in the presence of the fatty acid derivative 5-doxylstearic acid in

the oxidized form and after reduction with ascorbic acid (Battiste

and Wagner, 2000).

Isothermal Titration Microcalorimetry

Proteins were codialyzed against 100 mM potassium phosphate (pH

7.4) and 1 mM DTT. When appropriate, SCP2 was premixed with

stearoyl-CoA (Sigma) in a small molar excess, and 2 mM stearoyl-

CoA was added to the dialysis buffer to ensure uniform loading of

SCP2. Dialysates were degassed, and the concentration was mea-

sured by A280nm. ITC measurements were conducted on a MicroCal

VP-ITC using Pex5p(C) at 30–50 mM as a sample and SCP2 or PTS1

peptide (Sigma Genosys) at 350–750 mM 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 were 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 QuikChange 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). All primers used

are listed in Table S1. pPTS2-CAcT encoding an N-terminal PTS2

signal followed by the reporter protein CAcT was described previ-

ously (Braverman et al., 1998).

Human fibroblast cells were cultured at 37�C in Dulbecco’s mod-

ified 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 for 1 day on cov-

erslides in 60 mm tissue-culture dishes were transfected with

pPTS2-CAcT, pEGFP-SCP2, and one of the Pex5p expression plas-

mids using FuGENE 6 Transfection Reagent (Roche Diagnostics,

Mannheim, Germany). At various time points (24, 48, 72, and 96 hr

after transfection), cells were fixed with 3% formaldehyde in phos-

phate-buffered saline (PBS), permeabilized with 1% Triton X-100

in PBS, and subjected to immunofluorescence microscopy and

GFP fluorescence life imaging. Polyclonal rabbit antibodies against

CAcT and sheep antibodies against human catalase were pur-

chased from Invitrogen (Germany) and The Binding Site (UK), re-

spectively. 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 633/

1.4 oil objective and an Axiocam MR digital camera and were pro-

cessed with AxioVision 4.2 software (Zeiss, Jena, Germany).

Molecular Cell662

Supplemental Data

Supplemental Data include four figures and one table and can be

found with this article online at http://www.molecule.org/cgi/

content/full/24/5/653/DC1/.

Acknowledgments

We thank Ben Distel, Andre 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.S.) 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.

Received: March 21, 2006

Revised: August 16, 2006

Accepted: October 16, 2006

Published: December 7, 2006

References

Agne, B., Meindl, N.M., Niederhoff, K., Einwachter, H., Rehling, P.,

Sickmann, A., Meyer, H.E., Girzalsky, W., and Kunau, W.H. (2003).

Pex8p: an intraperoxisomal organizer of the peroxisomal import

machinery. Mol. Cell 11, 635–646.

Battiste, J.L., and Wagner, G. (2000). Utilization of site-directed spin

labeling and high-resolution heteronuclear nuclear magnetic re-

sonance for global fold determination of large proteins with limited

nuclear overhauser effect data. Biochemistry 39, 5355–5365.

Braverman, N., Dodt, G., Gould, S.J., and Valle, D. (1998). An isoform

of Pex5p, the human PTS1 receptor, is required for the import of

PTS2 proteins into peroxisomes. Hum. Mol. Genet. 7, 1195–1205.

Brocard, C.B., Jedeszko, C., Song, H.C., Terlecky, S.R., and Walton,

P.A. (2003). Protein structure and import into the peroxisomal

matrix. Traffic 4, 74–82.

Brown, N.P., Leroy, C., and Sander, C. (1998). MView: a web-com-

patible database search or multiple alignment viewer. Bioinfor-

matics 14, 380–381.

Bruenger, A., Adams, P., Clore, G., Delano, W., Gros, D., Grosse-

Kunstleve, R., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.,

et al. (1998). Crystallography & NMR system: a new software suite

for macromolecular structure determination. Acta Crystallogr.

D Biol. Crystallogr. 54, 905–921.

CCP4 (Collaborative Computational Project, Number 4) (1994). The

CCP4 suite: programs for protein crystallography. Acta Crystallogr.

D Biol. Crystallogr. 50, 760–763.

Choinowski, T., Hauser, H., and Piontek, K. (2000). Structure of sterol

carrier protein 2 at 1.8 A resolution reveals a hydrophobic tunnel

suitable for lipid binding. Biochemistry 39, 1897–1902.

Cliff, M.J., Williams, M.A., Brooke-Smith, J., Barford, D., and Lad-

bury, J.E. (2005). Molecular recognition via coupled folding and

binding in a TPR domain. J. Mol. Biol. 346, 717–732.

Conti, E., and Izaurralde, E. (2001). Nucleocytoplasmic transport

enters the atomic age. Curr. Opin. Cell Biol. 13, 310–319.

Costa-Rodrigues, J., Carvalho, A.F., Fransen, M., Hambruch, E.,

Schliebs, W., Sa-Miranda, C., and Azevedo, J.E. (2005). Pex5p, the

peroxisomal cycling receptor, is a monomeric non-globular protein.

J Biol Chem. 280, 24404–24411. Published online May 2, 2005. 10.

1074/jbc.M501985200.

Dammai, V., and Subramani, S. (2001). The human peroxisomal tar-

geting signal receptor, Pex5p, is translocated into the peroxisomal

matrix and recycled to the cytosol. Cell 105, 187–196.

D’Andrea, L.D., and Regan, L. (2003). TPR proteins: the versatile

helix. Trends Biochem. Sci. 28, 655–662.

Dodt, G., Braverman, N., Wong, C., Moser, A., Moser, H.W., Watkins,

P., Valle, D., and Gould, S.J. (1995). Mutations in the PTS1 receptor

gene, PXR1, define complementation group 2 of the peroxisome

biogenesis disorders. Nat. Genet. 9, 115–125.

Eichler, J., and Irihimovitch, V. (2003). Move it on over: getting pro-

teins across biological membranes. Bioessays 25, 1154–1157.

Erdmann, R., and Schliebs, W. (2005). Peroxisomal matrix protein

import: the transient pore model. Nat. Rev. Mol. Cell Biol. 6, 738–742.

Faber, K.N., van Dijk, R., Keizer-Gunnink, I., Koek, A., van der Klei,

I.J., and Veenhuis, M. (2002). Import of assembled PTS1 proteins

into peroxisomes of the yeast Hansenula polymorpha: yes and no!

Biochim. Biophys. Acta 1591, 157–162.

Frolov, A., Cho, T.H., Billheimer, J.T., and Schroeder, F. (1996). Ste-

rol carrier protein-2, a new fatty acyl coenzyme A-binding protein.

J. Biol. Chem. 271, 31878–31884.

Garcia, F.L., Szyperski, T., Dyer, J.H., Choinowski, T., Seedorf, U.,

Hauser, H., and Wuthrich, K. (2000). NMR structure of the sterol

carrier protein-2: implications for the biological role. J. Mol. Biol.

295, 595–603.

Gatto, G.J., Jr., Geisbrecht, B.V., Gould, S.J., and Berg, J.M. (2000).

Peroxisomal targeting signal-1 recognition by the TPR domains of

human PEX5. Nat. Struct. Biol. 7, 1091–1095.

Gould, S.J., and Collins, C.S. (2002). Opinion: peroxisomal-protein

import: is it really that complex? Nat. Rev. Mol. Cell Biol. 3, 382–389.

Gouveia, A.M., Guimaraes, C.P., Oliveira, M.E., Sa-Miranda, C., and

Azevedo, J.E. (2003). Insertion of Pex5p into the peroxisomal mem-

brane is cargo protein-dependent. J. Biol. Chem. 278, 4389–4392.

Holroyd, C., and Erdmann, R. (2001). Protein translocation machin-

eries of peroxisomes. FEBS Lett. 501, 6–10.

Huber, P.A., Birdsey, G.M., Lumb, M.J., Prowse, D.T., Perkins, T.J.,

Knight, D.R., and Danpure, C.J. (2005). Peroxisomal import of human

alanine:glyoxylate aminotransferase requires ancillary targeting

information remote from its C terminus. J Biol Chem. 280, 27111–

27120. Published online May 23, 2005. 10.1074/jbc.M502719200.

Jinek, M., Rehwinkel, J., Lazarus, B.D., Izaurralde, E., Hanover, J.A.,

and Conti, E. (2004). The superhelical TPR-repeat domain of O-linked

GlcNAc transferase exhibits structural similarities to importin alpha.

Nat. Struct. Mol. Biol. 11, 1001–1007.

Jones, T., Zou, J.-Y., Cowan, S., and Kjeldgaard, M. (1991). Im-

proved methods for building protein models in electron density

maps and the location of errors in these models. Acta Crystallogr.

A A46, 110–119.

Kabsch, W. (1988). Evaluation of single crystal x-ray diffraction data

from a position-sensitive detector. J. Appl. Crystallogr. 21, 916–924.

Kiel, J.A., Emmrich, K., Meyer, H.E., and Kunau, W.H. (2005). Ubiqui-

tination of the peroxisomal targeting signal type 1 receptor, Pex5p,

suggests the presence of a quality control mechanism during perox-

isomal matrix protein import. J Biol Chem. 280, 1921–1930. Pub-

lished online November 9, 2004. 10.1074/jbc.M403632200.

Klein, A.T., Barnett, P., Bottger, G., Konings, D., Tabak, H.F., and

Distel, B. (2001). Recognition of peroxisomal targeting signal type

1 by the import receptor Pex5p. J. Biol. Chem. 276, 15034–15041.

Knott, T.G., Birdsey, G.M., Sinclair, K.E., Gallagher, I.M., Purdue,

P.E., and Danpure, C.J. (2000). The peroxisomal targeting sequence

type 1 receptor, Pex5p, and the peroxisomal import efficiency of

alanine:glyoxylate aminotransferase. Biochem. J. 352, 409–418.

Krissinel, E., and Henrick, K. (2004). Secondary-structure matching

(SSM), a new tool for fast protein structure alignment in three dimen-

sions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268. Pub-

lished online November 26, 2004. 10.1107/S0907444904026460.

Kunau, W.H., Agne, B., and Girzalsky, W. (2001). The diversity of or-

ganelle protein import mechanisms. Trends Cell Biol. 11, 358–361.

Lametschwandtner, G., Brocard, C., Fransen, M., Van Veldhoven, P.,

Berger, J., and Hartig, A. (1998). The difference in recognition of ter-

minal tripeptides as peroxisomal targeting signal 1 between yeast

and human is due to different affinities of their receptor Pex5p to

the cognate signal and to residues adjacent to it. J. Biol. Chem.

273, 33635–33643.

Lamzin, V., and Wilson, K. (1993). Automated refinement of protein

models. Acta Crystallogr. D Biol. Crystallogr. 49, 129–147.

Target Recognition by the Import Receptor Pex5p663

Laskowski, R., MacArthur, M., and Thormton, J. (1993). PROCHECK:

a program to check the stereochemical quality of protein structures.

J. Appl. Crystallogr. 26, 283–291.

Lazarow, P.B. (2003). Peroxisome biogenesis: advances and conun-

drums. Curr. Opin. Cell Biol. 15, 489–497.

Lensink, M.F., Haapalainen, A.M., Hiltunen, J.K., Glumoff, T., and

Juffer, A.H. (2002). Response of SCP-2L domain of human MFE-2

to ligand removal: binding site closure and burial of peroxisomal

targeting signal. J. Mol. Biol. 323, 99–113.

Madrid, K.P., De Crescenzo, G., Wang, S., and Jardim, A. (2004).

Modulation of the Leishmania donovani peroxin 5 quaternary struc-

ture by peroxisomal targeting signal 1 ligands. Mol. Cell. Biol. 24,

7331–7344.

Matsuura, Y., and Stewart, M. (2004). Structural basis for the assem-

bly of a nuclear export complex. Nature 432, 872–877.

Modis, Y., Filppula, S.A., Novikov, D.K., Norledge, B., Hiltunen, J.K.,

and Wierenga, R.K. (1998). The crystal structure of dienoyl-CoA

isomerase at 1.5 A resolution reveals the importance of aspartate

and glutamate sidechains for catalysis. Structure 6, 957–970.

Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement

of macromolecular structures by the maximum-likelihood method.

Acta Crystallogr. D Biol. Crystallogr. 53, 240–255.

Neuberger, G., Maurer-Stroh, S., Eisenhaber, B., Hartig, A., and Ei-

senhaber, F. (2003). Motif refinement of the peroxisomal targeting

signal 1 and evaluation of taxon-specific differences. J. Mol. Biol.

328, 567–579.

Otera, H., Setoguchi, K., Hamasaki, M., Kumashiro, T., Shimizu, N.,

and Fujiki, Y. (2002). Peroxisomal targeting signal receptor Pex5p

interacts with cargoes and import machinery components in a spa-

tiotemporally differentiated manner: conserved Pex5p WXXXF/Y

motifs are critical for matrix protein import. Mol. Cell. Biol. 22,

1639–1655.

Parkes, J.A., Langer, S., Hartig, A., and Baker, A. (2003). PTS1-inde-

pendent targeting of isocitrate lyase to peroxisomes requires the

PTS1 receptor Pex5p. Mol. Membr. Biol. 20, 61–69.

Purdue, P.E., and Lazarow, P.B. (1996). Targeting of human catalase

to peroxisomes is dependent upon a novel COOH-terminal peroxi-

somal targeting sequence. J. Cell Biol. 134, 849–862.

Sattler, M., Schleucher, J., and Greisinger, C. (1999). Heteronuclear

multidimensional NMR experiments for the structure determination

of proteins in solution employing pulsed field gradients. Prog.

NMR Spectrosc. 34, 93–158.

Schafer, A., Kerssen, D., Veenhuis, M., Kunau, W.H., and Schliebs,

W. (2004). Functional similarity between the peroxisomal PTS2 re-

ceptor binding protein Pex18p and the N-terminal half of the PTS1

receptor Pex5p. Mol. Cell. Biol. 24, 8895–8906.

Schnell, D.J. (2000). Functions and origins of the chloroplast protein-

import machinery. Essays Biochem. 36, 47–59.

Seedorf, U., Raabe, M., Ellinghaus, P., Kannenberg, F., Fobker, M.,

Engel, T., Denis, S., Wouters, F., Wirtz, K.W., Wanders, et al.

(1998). Defective peroxisomal catabolism of branched fatty acyl

coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier

protein-x gene function. Genes Dev. 12, 1189–1201.

Shimozawa, N., Zhang, Z., Suzuki, Y., Imamura, A., Tsukamoto, T.,

Osumi, T., Fujiki, Y., Orii, T., Barth, P.G., Wanders, R.J., and Kondo,

N. (1999). Functional heterogeneity of C-terminal peroxisome target-

ing signal 1 in PEX5-defective patients. Biochem. Biophys. Res.

Commun. 262, 504–508.

Vagin, A., and Teplyakov, A. (1997). MOLREP: an automated pro-

gram for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025.

van der Klei, I., and Veenhuis, M. (2002). Peroxisomes: flexible and

dynamic organelles. Curr. Opin. Cell Biol. 14, 500–505.

Walton, P.A., Hill, P.E., and Subramani, S. (1995). Import of stably

folded proteins into peroxisomes. Mol. Biol. Cell 6, 675–683.

Wang, D., Visser, N.V., Veenhuis, M., and van der Klei, I.J. (2003).

Physical interactions of the peroxisomal targeting signal 1 receptor

pex5p, studied by fluorescence correlation spectroscopy. J. Biol.

Chem. 278, 43340–43345.

Weller, S., Gould, S.J., and Valle, D. (2003). Peroxisome biogenesis

disorders. Annu. Rev. Genomics Hum. Genet. 4, 165–211.

Wickner, W., and Schekman, R. (2005). Protein translocation across

biological membranes. Science 310, 1452–1456.

Winn, M.D., Isupov, M.N., and Murshudov, G.N. (2001). Use of TLS

parameters to model anisotropic displacements in macromolecular

refinement. Acta Crystallogr. D Biol. Crystallogr. 57, 122–133.

Yang, X., Purdue, P.E., and Lazarow, P.B. (2001). Eci1p uses a PTS1

to enter peroxisomes: either its own or that of a partner, Dci1p. Eur.

J. Cell Biol. 80, 126–138.

Accession Numbers

Coordinates and structure factors have been deposited at the

Protein Data Bank with accession codes 2C0L and 2C0M.