Topography for Independent Binding of α-Helical and PPII-Helical Ligands to a Peroxisomal SH3 Domain

Molecular Cell, Vol. 10, 1007–1017, November, 2002, Copyright 2002 by Cell Press

Topography for Independent Binding of ␣-Helical and PPII-Helical Ligands to a Peroxisomal SH3 Domain Alice Douangamath,1,5 Fabian V. Filipp,2,5 Andre´ T.J. Klein,3 Phil Barnett,3 Peijian Zou,1 Tineke Voorn-Brouwer,3 M. Cristina Vega,1 Olga M. Mayans,1 Michael Sattler,2,4 Ben Distel,3 and Matthias Wilmanns1,4 1 EMBL-Hamburg c/o Deutsches Elektronen Synchrotron Notkestrasse 85 D-22603 Hamburg 2 EMBL-Heidelberg Structural and Computational Biology Programme Meyerhofstrasse 1 D-69117 Heidelberg Germany 3 Academic Medical Center University of Amsterdam Department of Biochemistry P.O. Box 22700 1100 DE Amsterdam The Netherlands

Summary While the function of most small signaling domains is confined to binary ligand interactions, the peroxisomal Pex13p SH3 domain has the unique capacity of binding to two different ligands, Pex5p and Pex14p. We have used this domain as a model to decipher its structurally independent ligand binding sites. By the combined use of X-ray crystallography, NMR spectroscopy, and circular dichroism, we show that the two ligands bind in unrelated conformations to patches located at opposite surfaces of this SH3 domain. Mutations in the Pex13p SH3 domain that abolish interactions within the Pex13p-Pex5p interface specifically impair PTS1dependent protein import into yeast peroxisomes. Introduction Virtually all biological processes are governed by protein-protein interactions that are mediated by a limited set of modular domains, most of which are associated with one or more specific ligand binding preferences (Pawson and Scott, 1997). One of the most widely studied types of signaling modules is the Src homology 3 (SH3) domain (Kay et al., 2000; Mayer, 2001; Tong et al., 2002). Within the human genome, close to 500 SH3 domains have been identified to date, while less complex eukaryotes comprise a more limited set (29 in the S. cerevisiae genome). Despite the widespread occurrence of SH3 domains, their molecular function seems to be limited to stoichiometric single protein-protein interactions, either residing on the same polypeptide chain or 4

Correspondence: [email protected] (M.S.), wilmanns@ embl-hamburg.de (M.W.) 5 These authors contributed equally to this work.

via intermolecular interactions. Common to all known SH3 domains is a site between the RT and n-Src loops that generally binds a conserved PXXP sequence motif, which adopts a poly-proline type II (PPII) conformation. This motif is usually flanked by polar residues that determine its binding orientation. Since SH3 domains are involved in several signaling pathways regulating cell proliferation and differentiation, they have become conceptual targets for pharmacological intervention in a number of pathologies, such as different types of cancer. The current strategy in SH3directed drug discovery mostly focuses on the unique structure of the canonical SH3 PXXP binding site (Hiipakka et al., 2001) and its PXXP ligand binding motif. One of these approaches has led to the development of peptoidic and nonpeptoidic lead compounds (Nguyen et al., 1998, 2000), which may display antiproliferation activities when conjugated with cell-permeable vectors (Cussac et al., 1999). However, due to the abundance of SH3 domains and the limited variability of the PXXP binding site, it has been difficult to identify inhibitory molecules with single SH3 domain selectivity. Therefore, the discovery of a second ligand binding site could provide novel opportunities to target SH3 domains with multiple-site ligands (Hajduk et al., 1999). Pex13p is a membrane-bound central scaffold protein of the PTS1 signal-driven protein import into peroxisomes. In contrast to other known SH3 domains that are involved in mediating binary protein-protein interactions (Kay et al., 2000; Mayer, 2001), the C-terminal cytosolic Pex13p SH3 domain binds to at least two proteins, Pex5p and Pex14p, of the multiple-component PTS1receptor complex (Bottger et al., 2000; Girzalsky et al., 1999; Johnson et al., 2001; Urquhart et al., 2000). The three protein components may form a ternary complex, which could be tightened by direct interactions of the two Pex13p SH3 domains ligands, Pex5p and Pex14p (Albertini et al., 1997; Bottger et al., 2000). While binding of the Pex13p SH3 domain to Pex14p was shown to involve a classical PXXP-type interaction, the second ligand, Pex5p, is devoid of a recognizable PXXP motif, and hence, its interaction may be of a novel and as yet uncharacterized nature. Therefore, we chose this SH3 domain as a template to uncover the structural basis by which multiple ligand binding in this type of modular signaling domain may occur. By the combined use of X-ray crystallography and NMR spectroscopy, we show that Pex5p binds in an ␣-helical conformation to a novel site on the Pex13p SH3 domain that is structurally separated from the classical Pex14p binding site. Mutations in the Pex5p and Pex14p binding sites of the Pex13p SH3 domain show how binding of the two different ligands contributes to peroxisomal matrix protein targeting. While PTS1-dependent but not PTS2-dependent import into peroxisomes requires specific Pex13pPex5p interactions, both PTS1- and PTS2-dependent import requires Pex13p-Pex14p binding.

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Table 1. Crystallographic Statistics Crystal

Space Group

Unit Cell Dimensions a ⫽ 108.6 A˚, b ⫽ 50.6 A˚, c ⫽ 74.9 A˚, ␤ ⫽ 109.0 deg. a ⫽ 50.3 A˚, b ⫽ 63.2 A˚, c ⫽ 66.9 A˚

Pex13p(SH3) C2 Pex13p(SH3)-Pex14p(83–96) P212121 X-ray Data Collection

Pex13p(SH3)-peak Pex13p(SH3)-inflection Pex13p(SH3)-remote Pex13p(SH3)-Pex14p(83–96)

␭ (A˚)

d(min) (A˚) No. data

I/␴ Comp.a (%) Multiplicity (last shell) Rsymb (%)

Phasing powerc RCullisd

0.9785 0.9784 1.0446 1.1000

2.65 2.65 2.90 2.70

99.6 (100.0) 99.1 (99.9) 99.3 (100.0) 93.8 (97.4)

10,789 10,735 8,236 5,907

3.4 3.4 3.4 4.3

9.2 (4.5) 11.6 (3.6) 10.6 (3.7) 21.1 (2.9)

8.8 (22.1) 8.3 (29.2) 10.1 (35.5) 6.5 (33.0)

1.47 0.74 0.95 –

0.77 0.90 0.89 –

Solvent Ligand molecules residues

Rmsd bond length (A˚)

Rmsd bond angles (⬚)

Rcryste (%)

Rfreee (%) ⬍B⬎(A˚2)

66 11

0.007 0.007

1.35 1.31

23.5 23.4

27.7 30.8

Structure Refinement

Crystal

Protein (SH3) residues

Pex13p(SH3) 312 Pex13p(SH3)-Pex14p(83–96) 139

0 20

42.4 51.2

a

Completeness of unique data/Friedel pairs. Rsym ⫽ ⌺hkl⌺i|Ii(hkl) ⫺ ⬍I(hkl)⬎/⌺hkl⌺iIi(hkl). c Phasing power is defined as the ration of the rms value of the heavy atom structure factors amplitudes and the rms value of the lack-ofclosure error. Statistics are on acentric data and their anomalous values. d RCullis is the mean lack-of-closure error divided by the isomorphous/anomalous difference. Statistics are on acentric data and their anomalous values. e Rcryst and Rfree ⫽ ⌺|Fobs ⫺ Fcalc|/⌺Fobs; Rfree is calculated with 5% of the data that were not used for refinement. b

Results X-Ray Structure of the Unliganded Pex13p SH3 Domain To provide an accurate basis for molecular characterization of the two ligand binding sites, we solved the X-ray structure of an 86 residue construct of Pex13p comprising the SH3 domain at 2.65 A˚ resolution (Table 1). The structure reveals the compact, five-stranded ␤-barrel fold common to all known SH3 domains (Figures 1, 2A, and 2B). The N terminus of the structure contains an additional short 310 helix while the C terminus remained invisible. In comparison to most other SH3 domain sequences, the structure displays two insertions within the RT loop and the N-Src loop (Figure 1). Residues from these insertions provide an additional network of interactions that sandwich the aromatic side chain of Y361 that resides on strand ␤4, which is located at one of the flanks of the canonical SH3 domain PXXP binding site (Figure 2A). The structure suggests that this network may play a role in separating the binding sites for the two binding partners. Topography of Two Binding Sites for Pex5p and Pex14p Peptide Ligands We used heteronuclear NMR spectroscopy to characterize the Pex5p and Pex14p ligand binding sites (Figures 3–5). Changes in 1H and 15N chemical shifts were monitored during titrations of 15N-labeled Pex13p SH3 domain with synthetic peptides representing the previously identified binding motifs of Pex5p (residues 198– 214) and Pex14p (residues 83–96) proteins (Bottger et al., 2000; Girzalsky et al., 1999; Urquhart et al., 2000). Addition of either peptide induces large chemical shift

changes, which affect different amide groups (Figures 1, 4, and 5). Chemical shift perturbations induced by the Pex14p peptide are localized to the canonical PXXP binding site centered on strands ␤3 and ␤4 and involving the RT and N-Src loops and the 310 helix loop flanking this site. In contrast, addition of the Pex5p peptide induces chemical shift changes to the backbone amides at the N terminus, strands ␤1 and ␤2, the distal loop, and the RT loop. Thus, both binding sites are formed by similar sets of building blocks, a small antiparallel ␤ sheet segment (Pex5p, ␤1-␤2; Pex14p, ␤3-␤4) flanked by two or three loops. Because the Pex14p PXXP binding site is flanked by two large loops (RT, N-src) oriented toward this site, it appears as a deeper binding site cleft compared to the Pex5p binding site that is flanked by the short distal loop and the C terminus of the RT loop. Binding of Pex5p and Pex14p Peptide Ligands Is Independent The dissociation constants for the Pex13p SH3 domain interactions with Pex14p (residues 83–96) and Pex5p (residues 198–214) are 44 and 36 ␮M, respectively, as determined by NMR titration experiments. Thus, the binding affinities for both peptide ligands are within a range comparable to the ligand binding to other SH3 domains (Kay et al., 2000; Mayer, 2001). Except for two residues in the Pex13p-specific RT loop insertion (M324, V326), there is no overlap between the two binding sites. A surface representation of this SH3 domain shows that the two ligand binding sites are at opposite faces, suggesting independent binding (Figure 4). To test this hypothesis, two crosstitration NMR experiments were carried out in which the order of addition of Pex5p and Pex14p peptides was reversed (Figure 5A). In both ex-

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Figure 1. Structure-Sequence Relations in the Pex13p SH3 Domain The multisequence alignment includes presently known 29 SH3 domain sequences from S. cerevisiae. Except for the Pex13p SH3 domain sequence (top line), only the segments that belong to the canonical SH3 domain structure are included (Mayer, 2001). The locations of the secondary structural elements, as determined from the apo-Pexp13p SH3 domain crystal structure, are indicated above the alignment. The loops, connecting these elements, are labeled. Residues for which the accessible surface area dropped by more than 50% or whose backbone amides were considerably shifted (cf. Figures 4A and 4D) upon ligand binding are marked by “⫹”. Residues in the Pex14p binding site that are conserved among the SH3 domain sequences from S. cerevisiae are highlighted in magenta. None of the residues of the Pex5p binding site are conserved. Three residues (M324, P342, and Y361) forming a small structural cluster, absent in most other SH3 domains, are shown with orange background. Pex13p mutations (cf. Figures 5B and 6) are indicated on top.

periments, the same chemical shift changes and binding affinities were observed as in the single titration experiments, demonstrating that the two peptide binding sites are indeed independent and noncooperative. Pex5p and Pex14p Ligands Bind in Unrelated Conformations To gain direct structural insight into the conformations of the SH3 domain binding motifs of the two ligands, we used either X-ray crystallography when crystals became available or we applied NMR spectroscopy. Initial CD spectra of the isolated Pex14p peptide ligand indicated a PPII conformation, which does not change significantly when bound to the Pex13p SH3 domain (data not shown). However, the CD spectra of the Pex5p peptide ligand showed an increased ␣-helical content that is

induced upon binding to the Pex13p SH3 domain (Figure 3A). PP-Type II Conformation of Pex14p Ligand Binding Motif We were able to grow diffracting crystals of the Pex13p SH3 domain-Pex14p dodeca-peptide (83)EAMPPTLP HRDWK(96) complex allowing structure determination at 2.7 A˚ resolution. As indicated by CD spectroscopy, the peptide adopts a PPII conformation over a range of six residues (86–91) and binds to the canonical PXXP binding site in class II orientation (Figure 2B). This orientation is determined by the ligand C terminus where R93 (Pex14p) is salt bridged to E320 (Pex13p) and the indole side chain of W95 (Pex14p) is hydrogen bonded to E325 (Pex13p). The backbones of residues 89 and 90 (Pex14p) are also hydrogen bonded to N367 and Y368 of the Pex13p SH3 domain. Comparison of the NMR chemical

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Figure 2. Structures of the Pex13p SH3 Domain in the Absence and Presence of Peptide Ligands (A) Ribbon representation of the crystal structure of the apo-Pexp13 SH3 domain. The segments covering the canonical SH3 domain (Mayer, 2001) and the terminal extensions are shown in blue and cyan colors, respectively. The side chains of a network of three residues (M324, P342, and Y366), structurally bridging the RT loop and the n-Src loop, are shown in orange ball-and-sticks. The side chains of the conserved residues forming the canonical PXXP binding site in SH3 domains (2-3) (Y315, F317, W349, P363, and Y366; cf. Figure 1) are shown in magenta ball-and-sticks. The side chain atoms are displayed in atom-specific colors (carbon, black; oxygen, red; nitrogen, blue; sulfur, yellow). (B) Ribbon of the Pex13p(SH3)-Pex14p peptide complex structure. The Pex14p peptide is shown in yellow ball-and-sticks. In addition to the conserved residues in the PXXP binding site of the SH3 domain (cf. Figure 2A), residues E320 and E325, which provide specific interactions with side chains of the Pex14p peptide, are shown. (C) Conformation of the Pex5p peptide. Secondary structure defining NOEs in the Pex5p peptide when bound to the Pex13p SH3 domain, indicating ␣-helical conformation for residues 204–214 (cf. Figure 3B). The secondary structure is evidenced by strong H␣(i),HN(i⫹3), H␣(i), H␤(i⫹3), and sequential HN,HN NOE cross peaks (Wu¨thrich, 1986) in the filtered NOESY spectra. NOEs in the Cterminal region of the helix could not be unambiguously assigned due to signal overlap. (D) The ␣-helical region of the Pex5p peptide when bound to the Pex13p SH3 domain is shown as a cylinder. Positively charged, negatively charged, polar, and other residues are shown in blue, red, green, and gray shapes, respectively. Residues that are located on the front and back sides of the helix are in black and white circles, respectively.

shift perturbation data and changes of surface accessibility of the SH3 domain structure upon Pex14p ligand binding reveals the same binding site and shows that residues Y315, F317, R345, W349, and Y366 of Pex13p play critical roles in Pex14p association. Our data also provide direct structural rationale for loss of binding by mutations of E320 and W349 of the Pex13p SH3 domain (Bottger et al., 2000; Girzalsky et al., 1999). ␣-Helical Conformation of Pex5p Ligand Binding Motif Since no crystals of the Pex13p-Pex5p complex could be obtained, we characterized its secondary structure by NMR spectroscopy. Homonuclear proton TOCSY and NOESY experiments were used to assign the free Pex5p peptide, while heteronuclear 13C/15N-filtered TOCSY and NOESY experiments were recorded on a complex between 13C/15N-labeled Pex13p SH3 domain and unlabeled peptide to study the bound conformation. The data revealed an ␣-helical conformation of Pex5p over 2.5 turns (residues 204–214) when bound to Pex13p (Figures 2C, 2D, and 3B). Helical (i,i⫹3) NOE connectivities for residues 211–214 could not be unambiguously assigned due to signal overlap and, therefore, are not

indicated in Figure 2C. However, the H␣ secondary chemical shifts clearly indicate an ␣-helical conformation of these residues as well (Figure 3B). The NMR assignments suggest that the Pex5p helix does not extend N-terminally through P203 and is probably N capped by the negatively charged residues preceding this proline. Comparison of the H␣ secondary chemical shifts (⌬␦ H␣) of the Pex5p peptide bound versus unbound to Pex13p indicates a reduced ␣-helical content in the free Pex5p peptide ligand (Figure 3B). These data are consistent with our CD spectroscopy measurements (Figure 3A) and suggest induced folding of Pex5p upon Pex13p SH3 domain binding. Nonconserved but Specific Pex5p-Pex13p Interactions Are Essential for PTS1-Dependent Peroxisome Import To further investigate the molecular basis of Pex5p binding to the Pex13p SH3 domain and to assess the role of this interaction in PTS1-mediated protein import into peroxisomes, we created five Pex13p SH3 domain mutants in which Pex5p binding site residues were

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Figure 3. Increased ␣-Helical Content of Pex5p upon Pex13p SH3 Domain Binding (A) Conformation of the Pex5p peptide upon binding to the Pex13p SH3 domain by CD spectroscopy. The fraction of ␣-helical conformation of Pex5p peptide increases upon complex formation with Pex13p SH3 (curve in green color), whereas isolated Pex5p peptide remains mostly unstructured (curve in blue color). The curve in green represents the molar ellipticity difference between the spectrum of the complex (at a molar ratio of 2:1 Pex5p/Pex13p SH3) and the sum of the spectra of the free peptide and the SH3 domain. The distortion of the helical curve beyond 210 nm is due to the presence of aromatic residues in the sequence of Pex5p. (B) H␣ secondary chemical shifts for residues 198–216 of the Pex5p peptide in the free (blue) and bound form (green). Upon ligand binding, the ␣-helical content of the Pex5p peptide is increased, and the helix is extended beyond Lys210 up to Glu214.

changed. We selected three residues in central locations of the binding site mapped by NMR spectroscopy (cf. Figure 4), F310A (strand ␤1), L333A (strand ␤2), and A335Y (strand ␤2), and one double mutant (F310A, L333A). In a fifth mutant, the charges of two residues at the periphery of the Pex5p binding site were reversed (R353E, K355E). These two residues are located at the C terminus of strand ␤3 and the subsequent distal loop. All five Pex13p SH3 domain mutants showed 1H,15N correlation NMR spectra comparable to that of the wildtype structure, indicating that their tertiary structures are not changed (Figure 5B). These mutants were used first to measure binding of the Pex5p peptide in NMR titration experiments that monitor the 1H and 15N chemical shifts (Figure 5B). The

three Pex13p SH3 domain single mutants (F310A, L333A, and A335Y) showed at least 2-fold reduced affinity compared to that of the wild-type protein. In these mutants, complete or nearly complete binding of the Pex5p peptide requires 3- to 10-fold larger molar ratios of Pex5p-Pex13p than binding of Pex5p to the wild-type Pex13p SH3 domain. The two double mutants F310A/ L333A and R353E/K355E do not show any chemical shift changes, even at 4-fold molar excess of Pex5p peptide, indicating that the binding affinity is reduced at least 100-fold. Since all mutant proteins are structurally unchanged, we conclude that the mutated residues directly lower or impair binding to the Pex5p ligand. The two double mutants that showed the strongest effects bound Pex14p with comparable affinity as did the wildtype Pex13p SH3 domain (Figure 5B), reconfirming the independence of the Pex5p and Pex14p peptide binding sites (Figure 5A). To test the effects of these mutations on PTS1-dependent peroxisome import, we applied a previously developed in vivo assay in which cells with a defective PTS1dependent protein import show reduced growth on media with oleic acid as sole carbon source (Bottger et al., 2000; Barnett et al., 2000). As a control, we used the Pex13p (E320K) mutant that is specifically impaired in Pex14p binding and as a consequence exhibits a defect in PTS1 and PTS2 import leading to strongly reduced growth (Bottger et al., 2000; Girzalsky et al., 1999; Elgersma et al., 1996). A set of full-length PEX13 genes, containing the same mutations as those used for the NMR in vitro analysis, were generated and expressed in pex13⌬ cells under the control of the PEX13 promoter. As controls, pex13⌬ cells transformed with a plasmid expressing either wild-type Pex13p, Pex13p(E320K), or no insert (empty vector) were used. The growth rate on liquid oleate medium of pex13⌬ cells expressing any of the two double mutants (R353E/K355E; F310A/L333A) and the control mutant E320K were strongly reduced compared to that of wild-type Pex13p (Figure 6A). Two of the single mutants, F310A and A335Y, also showed a reduced growth rate, whereas the mutant L333A was less affected. All mutants grew on glucose or glycerol medium at rates comparable to those of the wild-type (data not shown). These results are in line with the NMR data and suggest critical roles for the mutated residues in contacting Pex5p. To further demonstrate the specificity of the sitedirected Pex13p mutants on PTS1-dependent import, they were coexpressed in pex13⌬ cells with either green fluorescent protein (GFP) fused to the tripeptide SKL (GFP-PTS1) to measure PTS1-dependent import or with GFP fused to 3-ketoacyl-CoA thiolase (PTS2-GFP) to measure PTS2-dependent import. Similarly, wild-type Pex13p, Pex13p (E320K), and empty vector were coexpressed in pex13⌬ cells with either GFP-PTS1 or PTS2GFP. GFP was visualized by direct fluorescence microscopy (Figure 6B). In pex13⌬ cells expressing wild-type Pex13p, GFP-PTS1 showed clear punctated fluorescence indicative of import of GFP-PTS1 into peroxisomes, whereas in pex13⌬ cells only cytosolic fluorescence was detected. Four of the Pex13p mutants (A335Y, F310A, F310A/L333A, and R353E/K355E) and the control mutant E320K revealed a diffuse, cytosolic fluorescence in addition to a weak punctated pattern

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Figure 4. Pex14p (Magenta, [A]–[C]) and Pex5p (Green, [D]–[F]) Binding Sites on the Pex13p SH3 Domain NMR chemical shift changes (⌬␦, as defined in the Experimental Procedures) in the presence of saturated concentrations of the Pex14p and Pex5p ligands are shown in (A) and (D), respectively. The topography of the Pex14p and Pex5 binding sites on the Pex13p SH3 domain is shown in surface (B and E) and ribbon (C and F) representations, respectively. The orientation of the Pex14p binding site in (B) and (C) is as in Figure 2A. Surface (E) and ribbon (F) representations of the Pex5p binding site are in an orientation rotated by 180⬚ around a vertical axis. Exposed side chains of residues for which large chemical shift perturbations were observed are shown in ball-and-stick presentation (C and F).

of labeling, suggesting partial mislocalization of GFPPTS1. GFP-PTS1 mislocalization was most prominent in the two double mutants (F310A/L333A and R353E/ K355E) and in E320K, which is in line with the observed strong growth defect of these mutants on oleate. In contrast, the distribution of the PTS2 reporter protein PTS2-GFP in the Pex5p binding site mutants was comparable to that of wild-type Pex13p, implying that in these mutants, PTS2-mediated import is unaffected. As reported previously, Pex13p (E320K), which is specifically affected in its interaction with Pex14p (Bottger et al., 2000; Girzalsky et al., 1999), impairs both PTS1- and PTS2-dependent protein import. The structural determination of the Pex5p binding site on the Pex13p SH3 domain has allowed us to probe, both in vitro and in vivo, the involvement of individual residues into Pex5p binding and, thus, in PTS1-dependent peroxisome import. Data from the NMR titration experiments and the PTS1 and PTS2 localization studies highly correlate and indicate that the molecular interactions within the Pex5p-Pex13p interface are determinants of PTS1-dependent import. Moreover, the reduced growth rates of these Pex5p binding site mutants further support the significance of the Pex13p-Pex5p interaction in vivo. In all experimental setups, the two double mutants (F310A/L333A and R353E/K355E) show the strongest effects, suggesting that abolishment of interactions of the single residues may not be sufficient to completely impair Pex5p-Pex13p binding.

Discussion Pex13p SH3 Domain as Multiple Ligand Scaffold Involved in Peroxisome Import Our data enable us for the first time to structurally characterize a unique SH3 domain that exhibits two independent, nonoverlapping ligand binding sites. The Pex5p ligand binds to a novel surface patch, located opposite to that of the Pex14p binding site. While the SH3 domain ligands characterized thus far share PPII conformation, the Pex5p ligand adopts an ␣-helical conformation upon Pex13p SH3 domain binding over at least 11 residues. In a previous suppressor mutation analysis, two residues from the RT loop (N321 and E323) and two residues from the distal loop (R353 and K355) of Pex13p were identified to be potentially involved in Pex5p binding, suggesting a binding site between the RT loop and the distal loop (Barnett et al., 2000). However, the Pex5p binding site, as characterized by the NMR titration experiments, is centered on the concave surface comprised of strands ␤1 and ␤2. The regions previously identified by Barnett et al. (2000) are supported by the NMR experiments—for instance, by a large chemical shift change for K355—but are situated at the periphery of the Pex5p binding site. In contrast, the NMR titration experiments do not support the notion that N321 and E323 are part of the Pex5p binding site, even though they are implicated by the suppressor mutation screen. Since these mutants all involve drastic residue changes

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Figure 5. NMR Titration Experiments Demonstrating Effects of Pex5p Site Mutations of the Pex13p SH3 Domain on Binding to Pex5p and Its Independence from the Pex14p Binding Site (A) Independent binding of the Pex14p and Pex5p ligands to the Pex13p SH3 domain. 1H,15N correlation NMR spectra are shown for the uncomplexed (black) and the simultaneously Pex14p and Pex5p bound SH3 domain. Titration endpoints where the order of peptide addition was reversed are shown in blue (addition of Pex5p, then Pex14p) and orange (addition of Pex14p, then Pex5p). Chemical shift changes induced by Pex14p and Pex5p are indicated by pink and green colors, respectively. (Inset) The titration endpoints of M334 and I362 are independent of the order of addition of the peptide ligands, confirming the independence of the two binding sites. The titration curves and corresponding binding affinities are also independent of the occupancy of the second site demonstrating that the formation of the ternary complex is noncooperative. (B) Pex5p site mutations of the Pex13p SH3 domain. The 3D structure of Pex13p SH3 mutants is not disrupted since 1H, 15N correlation spectra of the 15N-labeled mutants are comparable to those of the wild-type Pex13p SH3 domain. Backbone amide resonances in the proximity of the point mutations show slightly different relative positions (e.g., A311, M334, V352). Saturation points used for Pex5p titrations are: wt, 1:2; A335Y, 1:4; L333A, 1:3; F310A, 1:10; (F310A, L333A), 1:4 (no effect); (R353E, K355E), 1:4 (no effect). Reference spectra in the absence of peptide and protein-ligand complexes at equimolar concentrations are shown in black and blue contours, respectively. The chemical shift change upon Pex5p addition is traced by a green line to saturation. Green contours correspond to protein-peptide molar ratios at saturation of binding. For the two double mutants, no binding of the Pex5p peptide is observed even at 4-fold excess peptide. To demonstrate that Pex14p binding is not affected in the Pex13p mutants, the trace of the chemical shift change of Y361 upon Pex14p ligand binding as well as the saturation point are shown in magenta for the wild-type SH3 domain and the two double mutant SH3 domains. Saturation of Pex14p binding is achieved at a molar 1:2 protein-peptide ratio in the double mutants, which is comparable to the wild-type protein. The concentration of the wild-type and mutant proteins is 0.2 mM.

(N321I/Y, E323V, R353G), it is likely that indirect effects, including conformational changes within the respective loops, may lead to their suppression activity. Although under in vivo conditions cargo loading of the Pex5p receptor may modulate binding of the Pex5p and Pex14p ligands to Pex13p (Urquhart et al., 2000), our NMR crosstitration data show that the corresponding peptide ligands bind independently. While the Pex14p PXXP binding site is highly conserved among known SH3 domains (Figure 1), suggesting PPII ligand binding to be a common property of these domains, the Pex5p binding site is devoid of any conserved residues and, therefore, is likely to be specific to the Pex13p SH3 domain. From our data and that of others (Girzalsky et al., 1999; Bottger et al., 2000; Stein et al., 2002), a molecular model is emerging in which Pex13p has been designated to play a pivotal role in peroxisomal matrix protein import. Early in the import process, Pex13p function is

required for both PTS1- and PTS2-dependent import by recruiting the receptor docking protein Pex14p to the translocation complex via canonical SH3-PXXP interactions. In a subsequent step of the import process, most likely after receptor docking (Urquhart et al., 2000), the PTS1 and PTS2 pathways diverge and utilize different regions within Pex13p. While the noncanonical binding site on the SH3 domain is used for Pex5p interactions, there is recent evidence to suggest that the N terminus of Pex13p is involved in binding the PTS2 receptor Pex7p (Stein et al., 2002). Consequently, Pex13p mutations may either block both import routes or affect only one of them. SH3 Domains: More Than PXXP Ligand Binders? The interaction site for ␣-helical Pex5p is the first distinct intermolecular ligand binding site identified in an SH3 domain that is structurally separate from the canonical PXXP binding site and some variations thereof (Nishida et al., 2001). Although some SH3 domains also employ

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Figure 6. In Vivo Analysis of Mutants in the Pex5p Binding Site of Pex13p SH3 (A) pex13⌬ cells expressing wild-type Pex13p (open diamonds), Pex13p (E320K) (open squares), Pex13p (A335Y) (closed squares), Pex13p (L333A) (closed circles), Pex13p (F310A) (open triangles), Pex13p (R353E/K355E) (closed triangles), Pex13p (L333A/F310A) (crosses), or no insert (empty vector) (closed diamonds) were precultured in 0.3% minimal glucose medium and inocculated at OD600 of 0.05 in minimal oleate medium, and growth was followed with time by measuring the optical density at 600 nm. (B) The strains as described in (A) were cotransformed with either GFP-PTS1 or PTS2-GFP, cultured on liquid oleate medium, and examined under the fluorescence microscope to visualize the distribution of the GFP fusion protein.

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alternative surface patches, their role is restricted to the association with adjacent domains from the same polypeptide chain. Notably, the C-terminal strand ␤5 plays a role in the relatively loose SH2-SH3 domain interface formation of Src family protein kinases (Sicheri et al., 1997; Xu et al., 1997) and in interdomain ␤ sheet formation of PSD-95 family members (McGee et al., 2001; Tavares et al., 2001). Thus, the Pex13p SH3 domain may serve as a model to reveal the potential of SH3 domains to be involved in multiple unrelated interactions with ligands. Recent genomic approaches geared toward the quantitative identification of protein-protein interactions in specific organisms (Gavin et al., 2002; Ho et al., 2002) are suggesting that this may be a more general phenomenon than was previously anticipated, even for small signaling domains like the SH3 domains. For instance, a recent analysis using a combined approach of phage display and yeast two-hybrid screens showed that many SH3 domains may be involved in multiple ligand interactions (Tong et al., 2002). To what extent these interactions are competitive or independent, however, still remains to be analyzed in further detail. In summary, the concept of multiple and independent ligand binding introduces a key element for the differential and regulated involvement of these domains in biological processes. Within the pharmacological perspective, these findings may provide a starting point for the development of multiple-site lead compounds targeted to modular SH3 domains that are central to many pathways in cell proliferation and differentiation. Experimental Procedures Sample Preparation for Structural Analysis The plasmid pMALc2-Pex13p(SH3), encoding the maltose binding protein fused to Pex13p from S. cerevisiae (residues 301–386), was transformed in the methionine auxotrophic E. coli strain B834(DE3). The cells were grown at 37⬚C in methionine-deficient medium supplemented with 50 mg per liter of selenomethionine. When OD600 reached a value of 0.5, the expression of the recombinant protein was induced by the addition of 1 mM IPTG. The cells were further grown to an OD600 of 1.2 and harvested by centrifugation. Pex13p(SH3) was eluted from an amylose affinity chromatography column with 20 mM maltose and 200 mM NaCl in 50 mM Tris buffer (pH 8.0), followed by cleavage of the fusion protein by factor Xa protease. The cleavage products were passed over a size exclusion chromatography column (Superdex 75). The pure protein was concentrated to 15 mg/ml. For the binding site mapping, 15N- and 13C,15N-labeled Pex13p SH3 domain (residues 303–373) was expressed from a modified pET24d expression vector as GST fusion with an N-terminal six histidine tag and a TEV protease cleavage site between the GST tag and the SH3 domain. Transformants of the E. coli BL21(DE3) pLysS strain were grown at 37⬚C in a minimal medium containing 0.5g/l of 15 N-ammonium chloride and 10% (w/v) 13C glucose. Sonicated cell lysates were passed through a metal-chelate resin preloaded with Ni2⫹. The fusion protein was eluted in the presence of 330 mM imidazole, in 20 mM Tris buffer (pH 8.0) and 150 mM NaCl. The eluate was digested with TEV protease in 5 mM Tris buffer (pH 8.0) and 100 mM NaCl. Protease and residual undigested protein were removed using a second metal-chelate resin step. The digested protein was loaded onto a size exclusion chromatography column (Superdex75) equilibrated in 20 mM sodium phosphate buffer (pH 6.5) and 80 mM NaCl. The Pex13p SH3 domain eluted at a volume of 95 ml and was concentrated to a maximum of 0.6 mM. Plasmid DNA of Pex13p SH3 domains containing site-directed mutations were amplified and cloned from pET24d-(His)6-GST-TEVPex13p(SH3) DNA. 15N-labeled Pex13p mutants were expressed and purified as wild-type protein.

The peptides used comprise residues 198–214 (EQEQQPWTDQ FEKLEKE) and residues 198–216 (EQEQQPWTDQFEKLEKEVS) of Pex5p and residues 83–96 (EAMPPTLPHRDWKD) of Pex14p. The longer Pex5p peptide was used to investigate whether V215 may also be required for the Pex13p interaction. However, this is not the case, since in the NMR titrations of Pex13p, both Pex5p peptides yielded similar 1H,15N-correlation spectra and titration curves, indicating identical binding. X-Ray Structure Determination of the apo-Pexp13 SH3 Domain Crystals of the apo-Pex13p SH3 domain were grown within 3 days by mixing equal volumes of the protein and the reservoir solution (1 ␮l each), followed by equilibration against the reservoir solution containing 2.2 M ammonium sulfate, 0.1 M sodium phosphate, 100 mM Bis-Tris-Propane (pH 8.0), and 2% (v/v) ethanol at 22⬚C. A crystal of approximate size of 150 ⫻ 60 ⫻ 5 ␮m3 was transferred from the original sitting drop to a cryoprotectant solution containing 23% (v/v) glycerol and flash frozen in liquid nitrogen before being irradiated to the X-ray beam in a nitrogen cryostream. A multiple anomalous dispersion dataset was collected at ESRF, Grenoble, France (beamline ID14-4) at 3 wavelengths, corresponding to the peak, the inflection, and a high-energy remote point on the selenium fluorescence spectrum. The data were processed with the Denzo and Scalepack software packages (Otwinowski and Minor, 1997). Six out of eight seleno-methionine positions (four molecules per asymmetric unit with two seleno-methionine sites each) were found by SHELXS (Sheldrick et al., 1993) and refined with SHARP (LaFortelle et al., 1997). One of the two NCS axes was found using FINDNCS (Lu, 1999) and located within the asymmetric unit using GETAX (Vonrhein and Schulz, 1999). A molecular mask was created using MAMA software (Kleywegt and Jones, 1999) and provided the basis for 2-fold averaging by DM (Cowtan and Main, 1998). The resulting electron density map was of sufficient quality to subsequently identify the second NCS axis, to build the model using O (Jones et al., 1991), and to refine it to 2.65 A˚ resolution using CNS (Brunger et al., 1998). The final model includes residues 301–373 (chain A), 301–377 (chain B), 301–320, 323–371 (chain C), 301–372 (chain D), and 66 ordered solvent molecules. The final R factor and Rfree of this model are 23.5% and 27.7%, respectively. Further statistics are given in Table 1. X-Ray Structure Determination of the Pex13p-Pex14p Complex Pex13p(SH3) at 1.25 mM was mixed with 5 mM peptide EAMPPTLPHRDWKD (residues 83–96 of Pex14p). Crystals of the complex Pexp13(SH3)-Pex14p(83–96) were obtained at 22⬚C by vapor diffusion with a reservoir containing 2.0 M ammonium sulfate, 100 mM Tris (pH 7.3), and 200 mM lithium sulfate. A crystal of approximately 150 ⫻ 50 ⫻ 50 ␮m3 was flash frozen on the X-ray beam without addition of further cryoprotectant. Data were collected to 2.7 A˚ resolution at EMBL-Hamburg, DESY, Germany (beamline X31) and processed with the DENZO and SCALEPACK software packages (Kleywegt and Jones, 1999). Phases were obtained by using the apo-Pex13p(SH3) coordinates as the search model for molecular replacement with the program BEAST (Read, 2001). The structure was refined with REFMAC5 (Murshudov et al., 1999) to a final R factor and Rfree of 23.8% and 30.4%, respectively. The final model includes two Pex13p-Pex14p complexes, in which residues 301–372 and 305–371 of the Pex13p molecules and residues 86–95 of each of the two Pex14p molecules are defined. NMR Spectroscopy Chemical shift assignments were obtained using standard homoand heteronuclear experiments (Sattler et al., 1999). Heteronuclear multidimensional NMR experiments were recorded on a 15N, 13 C-labeled Pex13p SH3 domain. Chemical shift changes (⌬␦ ⫽ {(⌬␦1H)2 ⫹ (⌬␦15N)2}1/2, in ppm) during the NMR titrations with the two peptides were monitored in two-dimensional 1H,15N correlation experiments recorded on a 0.25 mM solution of 15N-labeled SH3 domain. A complete histogram presentation of the chemical shift changes is provided in Figures 4A and 4D. The titration curves (measured at nine ligand concentrations) were fitted assuming a bimolecular binding event as described (Liu et al., 1999). Dissociation constants of 44 ⫾ 40 ␮M and 36 ⫾ 25 ␮M were obtained

Molecular Cell 1016

from analyzing 26 and 25 amide signals for the Pex14p and Pex5p interaction, respectively. Circular Dichroism Stock peptide solutions for CD experiments were prepared in 20 mM sodium phosphate buffer (pH 7.5). The concentration of peptide samples was determined by their absorbance at 280 nm in the presence of denaturing agent. The CD experiments were performed with a 0.2 cm path-length cell, using a Jasco-710 instrument calibrated with (1S)-(⫹)-10-camphorsulphonic acid. Reported spectra are the average of 20 scans performed in the range 200–250 nm at 20⬚C by taking points every 0.2 nm, with 100 nm min⫺1 scan rate, 1 s integration time, and 1 nm bandwidth. The binding experiments were carried out for a 20 ␮M Pex13 SH3 solution in 20 mM sodium phosphate (pH 7.5) at increasing concentrations of Pexp5 peptide. Yeast Strains and Culture Conditions For in vivo analysis the yeast strain BJ1991pex13⌬ (MAT␣, pex13::LEU2, leu2, trp1 ura3-251, prb1-1122, pep4-3, gal2) was used (Elgersma et al., 1996). Yeast transformations and selection of transformants were carried out as described previously (Barnett et al., 2000). To follow growth on oleate, yeast cells were precultured in 0.3% glucose medium (0.3% glucose, 0.67% yeast nitrogen base without amino acids [YNB-WO: Difco] and amino acids [20–30 ␮g/ml] as needed) and inoculated in minimal oleate medium (0.1% oleic acid, 0.2% Tween-40, 0.1% yeast extract, 0.67% YNB-WO, and amino acids as needed) at an OD600 of 0.05. Plasmids and Cloning Procedures for In Vivo Analysis Yeast expression plasmids used in this study were: 20.46 (PEX13 under the control of he PEX13 promoter on a single copy plasmid) (Elgersma et al., 1996), 20.54 (pex13 [E320K] under the control of the PEX13 promoter) (Elgersma et al., 1996), and GFP-PTS1 (SKL fused to the C terminus of green fluorescent protein) (Bottger et al., 2000). PTS2-GFP was created by inserting the fragment encoding 3-ketoacyl-CoA thiolase fused to the N terminus of green fluorescent protein of plasmid Sc/thiolase-GFP (generous gift of Stephen Gould, Baltimore, MD) between the KpnI and XbaI sites of pEW156 (Hoepfner et al., 2001). Site Mutagenesis Site-directed mutants were generated using the Quick Change mutagenesis kit (Stratagene). Primers for mutations were designed following the manufacturer’s instructions. For in vivo analysis, the plasmid 20.46 expressing the wild-type PEX13 gene under the control of the PEX13 promoter and, for in vitro analysis, the wild-type Pex13p SH3 domain (residues 303–373) construct used for NMR experiments were used as the template for mutagenesis. The double mutant L333A/F310A was created in two steps. In the first mutagenesis round, the wild-type constructs were used as a template to generate the L333A mutation. Next, L333A mutants were used as a template to introduce the F310A substitution. All site-directed mutants were sequenced to confirm the presence of the desired mutation. Fluorescence Microscopy Transformants were grown on 0.3% glucose medium and shifted to minimal oleate medium for 8 hr. Cells were examined under a Zeiss Axiophot2 microscope, and images were captured by a Coolpix HQ CCD camera (Roper Scientific). All images were processed identically using Adobe Photoshop. Acknowledgments We would like to thank Katja Schirwitz and Gunter Stier for advice with protein expression and purification. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to M.W. (WI 1058/4-1) and by the EU Biotechnology Programme to B.D. and M.W. (BIO-CT97-2180). This contribution is dedicated to the memory of Matti Saraste. Received: May 23, 2002 Revised: October 16, 2002

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Accession Numbers

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Coordinates and structure factors for the Pex13p SH3 domain and the complex with the Pex14p ligand have been deposited in the Protein Data Bank, accession numbers 1JQQ and 1N5Z, respectively. Note Added in Proof While this manuscript was under review, we became aware of a study by Kami et al. (EMBO J. 21, 4268–4276, 2002) revealing a similar Pex5p binding site on the Pex13p SH3 domain. For this analysis, these authors used a peptide lacking the N-terminal residues (198–202) of the Pex5p ligand we used. The estimated binding affinity of the peptide used by Kami et al. was about 1 mM while ours measured at least 20 times higher binding affinity. Some of our mutations, showing strong in vivo effects (F310A), are outside the binding site identified by Kami et al., providing a potential structural rationale for the observed differences in binding affinities.