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Dec 28, 2006 - 29–253)-Elongin BC complex (PDB code. 2FNJ) ..... The ribbon drawing and surface representation of PSD (PDB code 2FBE) are in the same ...
Molecular Cell 24, 967–976, December 28, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.molcel.2006.11.009

Structural Basis for Protein Recognition by B30.2/SPRY Domains Jae-Sung Woo,1 Hye-Young Suh,1 Sam-Yong Park,2 and Byung-Ha Oh1,* 1 Center for Biomolecular Recognition Department of Life Sciences Division of Molecular and Life Sciences Pohang University of Science and Technology Pohang, Kyungbuk, 790-784 Korea 2 Protein Design Laboratory Yokohama City University Suehiro 1-7-29, Tsurumi-ku Yokohama 230-0045 Japan

Summary B30.2/SPRY domains are found in numerous proteins that cover a wide spectrum of biological functions, including regulation of cytokine signaling and innate retroviral restriction. Herein, we report the crystal structure of the B30.2/SPRY domain of a SPRY domaincontaining SOCS box (SSB) protein, GUSTAVUS, complexed with a 20 amino acid peptide derived from the RNA helicase VASA, revealing how these domains recognize target proteins. The peptide-binding site is conformationally rigid and has a preformed pocket. The interaction between the pocket and the Asp-Ile-AsnAsn-Asn-Asn sequence within the peptide accounts for the high-affinity binding between GUSTAVUS and VASA. This observation led to a facile identification of the Glu-Leu-Asn-Asn-Asn-Leu sequence as the recognition motif in a proapoptotic protein Par-4 for its interaction with a GUSTAVUS homolog, SSB-1. Ensuing analyses indicated that many B30.2/SPRY domains have a similar preformed pocket, which would allow them to bind multiple targets. Introduction The B30.2 domain (w190 amino acids) and the SPRY domain (w140 amino acids) were contemporaneously identified and thought to be unrelated (Ponting et al., 1997; Vernet et al., 1993). However, as the number of the proteins containing these domains expanded, it became apparent that the two domains share limited but detectable sequence homology. Notably, w53% of the SPRY domains are associated with a domain adjacent to their N terminus, termed PRY domain (50–60 amino acids). The confusing relationship between these domains was clarified by the elucidation of the structures of Drosophila GUSTAVUS/CG2944-PF (Woo et al., 2006) and the protein encoded by a human gene 19q13.4.1 (EST number CA454993) (Grutter et al., 2006), the former possessing a B30.2 or SPRY domain depending on classification and the latter composed of tandem PRY-SPRY domains. Both structures consist of a single-domain b sandwich fold with one or two a he*Correspondence: [email protected]

lixes. They are closely similar to each other despite no detectable sequence homology between the N-terminal 60 residue segments of the two proteins, supporting the idea that B30.2 and SPRY domains have derived from the same ancestral gene, and the first part of the gene encoding w60 residues has diversified greatly beyond a limit of the sequence similarity detection (Woo et al., 2006). This is the basis for our designation of these domains collectively as ‘‘B30.2/SPRY domains.’’ The B30.2/SPRY domain is present in more than 150 human proteins that can be classified into eleven protein families (Rhodes et al., 2005), including the SPRY domain-containing SOCS box (SSB) and tripartite/RBCC motif (TRIM) families. The SSB family proteins contain a central B30.2/SPRY domain and a C-terminal suppressor of cytokine signaling (SOCS) box. The SSB family proteins associate with Elongin C through the BC box in the C-terminal SOCS box, and each of SSB-1, -2, and -4 was shown to form a multiprotein E3 ubiquitin ligase complex with Elongin B, Elongin C, Cul5, and a RING protein (Kamura et al., 2004). Like many SOCS box-containing proteins, the SSB family proteins could function as receptors for the substrate proteins that are ubiquitinated by cullin-based ubiquitin ligases (Kile et al., 2002). GUSTAVUS is the only SSB family protein in Drosophila, and its interaction with VASA plays a critical role in oocyte development (Styhler et al., 2002). The TRIM family comprises a large number of proteins containing the tripartite/RBCC motif (RING domain, B-box, and coiled-coil region), and more than half of these proteins have an additional C-terminal B30.2/SPRY domain. The functional significance of the B30.2/SPRY domain in the TRIM family proteins is clear from the identification of many disease-causing point mutations in this domain of Pyrin and MID1 that are responsible for familial Mediterranean fever (FMF) and Opitz syndrome, respectively (Bakkaloglu, 2003; Schweiger and Schneider, 2003). Furthermore, the length polymorphism and/or speciesspecific residue changes that determine the specificity in retroviral restriction by the TRIM5a proteins are solely found in the B30.2/SPRY domain of these proteins (Song et al., 2005a, 2005b). Increasing evidence has implicated the B30.2/SPRY domains as protein-interacting modules. Nonetheless, the identity of target proteins recognized by the B30.2/ SPRY domains is known for only a few proteins, and, furthermore, the mechanism underlying the recognition of specific amino acid sequences by these domains is totally unknown. In this study, we determined the crystal structure of the B30.2/SPRY domain of GUSTAVUS in complex with a 20 residue VASA peptide, revealing that the peptide-binding site is conformationally rigid and has a preformed pocket. The pocket, interacting with a short segment within the peptide, plays a critical role in the high-affinity interaction of GUSTAVUS with VASA. Notably, the peptide-binding residues of GUSTAVUS are conserved in three human SSB family proteins and also in SPRY domain-containing F box (F box-SPRY) proteins. These observations led to the identification of a short sequence motif in prostate apoptosis response-4

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(Par-4) recognized by SSB-1 and imply that the SSB and F box-SPRY proteins recognize and regulate multiple target proteins, rather than a single target protein. In addition, the study suggests that peptide library-driven selection of short recognition sequences is a promising experimental approach to identify target proteins of the B30.2/SPRY domain-containing proteins. Results and Discussion Overall Structure The 20 amino acid synthetic peptide used in this study, which we designate as peptide I, is identical to residues 184–203 of VASA. Previously, we reported that fulllength VASA interacted with GUSTAVUS with an apparent dissociation constant (KD) of 40 nM, and a 30 amino acid stretch of VASA (residues 174–203) retained this strong binding affinity (Woo et al., 2006). By further truncation, we found that peptide I also bound to the GUSTAVUS protein as tightly as the 30 amino acid VASA peptide. Subsequently, we obtained the crystals of the B30.2/SPRY domain of GUSTAVUS (residues 29–234) in complex with peptide I, and the structure of the complex was determined to 2.2 A˚ resolution (Table 1). The peptide lies on a surface located at one end of the b sandwich core of the domain in a rather extended conformation (Figure 1A). The peptide-binding interface coincides with a surface on which we previously mapped the sequence variations in TRIM5a responsible for retroviral restriction and the FMF-causing point mutations in Pyrin (Woo et al., 2006). The interface, which we designate as surface A, is a buried area of 695 A˚2 constituted by five loops (Figure 1A; loop A to loop E). Spatially, surface A is distinctively opposite to the location of N and C termini of the domain that are close to each other. A superposition of the presented structure with that of GUSTAVUS (residues 29–253)-Elongin B-Elongin C complex shows that the bound peptide is on the opposite side of the complex of Elongins B and C (Elongin BC), with the B30.2/SPRY domain in between the two (Figure 1B). In this arrangement, VASA is presumed to bind GUSTAVUS independently of Elongin BC, which interacts with the C-terminal BC box of GUSTAVUS. Preformed Ligand-Binding Surface of GUSTAVUS The asymmetric unit of the crystal contained two molecules of the protein-peptide complex that exhibit largely different crystal-packing interactions; e.g., the bound peptide in one molecular complex is in contact with an adjacent molecule, but the peptide in the other complex is free of crystal-packing interaction. Nevertheless, the conformation of loop A to loop E is nearly the same between the two molecules of GUSTAVUS. The main-chain atoms of the five loops (26 residues) in the two molecules can be superimposed with an rmsd of 0.19 A˚. Furthermore, the five loops can be superimposed on those in peptide-unbound GUSTAVUS with an rmsd of only 0.22 A˚ for the main-chain atoms and 0.61 A˚ for all atoms (Figure 1B), clearly indicating that these loops are conformationally rigid and do not undergo a so called induced-fit conformational change upon binding of the peptide. The observed conformational rigidity appears to stem from the intricate intramolecular interactions

Table 1. Data Collection and Structure Refinement Statistics Data Collection Space group Unit cell dimensions a, b, c (A˚) Wavelength (A˚) Resolution (A˚) Rsyma I/s(I) Completeness (%) Redundancy

P41212 80.21, 80.21, 159.80 1.0000 50.0–2.2 3.6 (22.4)b 37.1 (3.6) 96.2 (79.9) 6.6

Refinement Resolution (A˚) Number of reflections Rworkc / Rfree Number of atoms Protein Peptide Water Rms deviations Bond lengths (A˚) Bond angles ( ) Average B values (A˚2) Protein Peptide Water

20–2.2 26,272 21.9 / 26.0 3160 301 142 0.0059 1.2619 44.2 67.7 44.0

a Rsym = SjIobs 2 Iavgj/Iobs, where Iobs is the observed intensity of individual reflection and Iavg is average over symmetry equivalents. b The numbers in parentheses are statistics from the highest resolution shell. c Rwork = SjjFoj 2 jFcjj/S jFoj, where jFoj and jFcj are the observed and calculated structure factor amplitudes, respectively. Rfree was calculated with 5% of the data.

that these loops are engaged in (see Figure S1 in the Supplemental Data available with this article online). A Prominent Pocket Is Primarily Responsible for the Binding Affinity An easily recognizable pocket is present on surface A of GUSTAVUS, whose wall is lined partly by the side chains of Tyr133 and Trp221 (Figure 2A). The pocket interacts with the N-terminal six residues of peptide I (Asp184Ile185-Asn186-Asn187-Asn188-Asn189; denoted as DI-N-N-N-N). The side chains of Asn186 and Asn188 are snugly and completely inserted into the pocket and engage in a total of six buried hydrogen bonds involving Tyr133 and other residues (Arg81, Thr115, Val220, and Gly222) of the protein (Figures 2A and 2B). In addition, the side chain of Asn186 is involved in an amino-aromatic interaction with Tyr133. Furthermore, Asn186 and Asn187 are involved in tight hydrophobic interactions with Trp221. These interactions explain why the substitution of Tyr133 with alanine or Trp221 with leucine severely affected the binding of the 30 amino acid VASA peptide (Woo et al., 2006). In addition to the three asparagine residues, Asp184, Ile185, and Asn189 are also involved in three hydrogen-bonding or hydrophobic interactions with the residues at the rim of the pocket (Figure 2A). Other than the D-I-N-N-N-N stretch, the C-terminal five residues of peptide I (Asp194, Val195, Glu196, Arg199, and Tyr202) interact with the protein, but not as extensively as the N-terminal residues (Figure 2A). Remarkably, the remaining nine residues of peptide I are separated from the protein atoms by

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Figure 1. Structures of GUSTAVUS (A) B30.2/SPRY domain in complex with peptide I. Loop A to loop E (blue), extending from the two b sheets (green and orange), constitutes the peptide-binding site. Peptide I (yellow) is shown as a coil for the main chain and sticks for the side chains. The secondary structural elements of the domain are sequentially labeled. The domain organizations of GUSTAVUS and VASA are shown. The regions in green and yellow on the diagrams indicate the fragments of the proteins used for the structure determination. (B) Structural superposition. The GUSTAVUS (residues 29–234)-peptide I complex is superposed on the GUSTAVUS (residues 29–253)-Elongin BC complex (PDB code 2FNJ), using the program SuperPose (Maiti et al., 2004). The B30.2/SPRY domain and peptide I are colored as in (A), and the BC box of GUSTAVUS is in magenta. The two superposed B30.2/SPRY domains are hardly discernible due to a very small structural difference between the two complexes.

>3.6 A˚, indicating that these residues do not interact or poorly interact with the protein. In order to address whether the D-I-N-N-N-N sequence of VASA is the key determinant for the interaction with GUSTAVUS, we prepared a short peptide identical to the six-residue sequence and a mutant protein of full-length VASA in which the three asparagines (residues 186–188) were substituted with alanines. By isothermal titration calorimetry (ITC), the binding affinity of the six-residue peptide for GUSTAVUS was found to be slightly better than that of peptide I (Figure 2C; KD of 23 nM), whereas that of the mutated VASA protein for GUSTAVUS was too low to determine the KD value by this method. These data indicate that the interaction between the prominent

pocket of GUSTAVUS and the D-I-N-N-N-N sequence of VASA is fully responsible for the high-affinity binding between the two proteins. Among the six residues of the D-I-N-N-N-N sequence, Asn186, Asn187, and Asn188 are nearly buried in the pocket (Figures 2A and 2B) and appeared to be the most important contributors to the binding affinity. In contrast, Asn189 is largely exposed to the bulk solvent, and only the backbone nitrogen atom of the residue makes a direct hydrogen bond with the protein (Figure 2A), suggesting that the substitution of this residue with any amino acid would have no or little effect on the binding affinity. For experimental verification, we prepared six mutants of peptide I: five containing a single substitution of D184A, I185A, N187A, N188A,

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Figure 2. Interaction of GUSTAVUS with Peptide I (A) A detailed view. The B30.2/SPRY domain is shown as a ribbon drawing in the transparent surface. The residues involved in the intermolecular interactions between peptide I (yellow) and the domain (white) are represented as sticks. Labeled are loop A to loop E and the residues involved in the intermolecular interactions. Dotted lines indicate the intermolecular hydrogen bonds. A red sphere indicates a bound water molecule. (B) Shape complementarity. The interaction of the protein (mesh model) with peptide I (CPK model) is superficial except for the pocket that accepts the side chains of Asn186, Asn187, and Asn188 (not visible). Ile185 caps the interaction between Asn186 and the pocket. (C) ITC analysis. The interactions between the B30.2/SPRY domain of GUSTAVUS and the indicated VASA peptides were analyzed by ITC, and the measured KD values are shown.

or N189A and the remaining one containing double substitutions of N186A and N189A. The doubly mutated peptide was synthesized instead of a peptide containing a single N186A mutation that was refractory to the synthesis. Compared with the wild-type peptide I, the peptides containing the N187A or N188A substitution interacted with the protein with drastically decreased binding affinities (Figure 2C; >30-fold increased KD). The D184A or I185A substitution also decreased the affinity, but much less significantly (Figure 2C; 5- to 7-fold increased KD). While the N189A substitution had little effect on the affinity, the peptide containing both the N186A and N189A substitutions exhibited drastically decreased affinity (Figure 2C; w800-fold increased KD), indicating that Asn186 is indeed an important contributor to the binding affinity. Therefore, we concluded that the preformed pocket interacts tightly with the six-residue sequence of D-I-N-N-N-X, where X represents any amino acid.

Identification of a Par-4 Segment that Interacts Strongly with SSB-1 The B30.2/SPRY domain of GUSTAVUS exhibits medium to high sequence homology with those of human SSB family proteins: 76%, 54%, 22%, and 75% sequence identity with SSB-1, -2, -3, and -4, respectively. According to the sequence alignment (Figure 3A), all the peptide I-interacting residues of GUSTAVUS, including the pocket-lining residues, are identically conserved in SSB-1 and SSB-4. This observation strongly suggests that a prominent pocket is present on surface A of the two proteins and that it accepts the N-N-N sequence motif. Recently, endogenous Par-4 was shown to bind SSB-1, SSB-2, and SSB-4, when each of the recombinant SSB proteins was expressed in 293T cells (Masters et al., 2006). Par-4 is a proapoptotic protein upregulated in prostate cancer cells and neuronal cells undergoing apoptosis (El-Guendy and Rangnekar, 2003; Gurumurthy and Rangnekar, 2004). Human Par-4 shares no

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Figure 3. Interaction of Par-4 Peptides with the SSB Family Members (A) Sequence alignment. The red, pink, and gray columns indicate the residues similarly conserved in five, four, and three of the aligned proteins, respectively. The secondary structural elements of GUSTAVUS are shown at the top of the alignment. The blue lines and labels indicate loop A to loop E. The triangles indicate the peptide I-interacting residues of GUSTAVUS. Of these, the pocket-forming residues are indicated by the filled triangles. (B) E-L-N-N-N-L sequence motif in Par-4. The motif is indicated by the green bar on the diagram for the primary structure of Par-4. ‘‘SAC’’ and ‘‘LZ’’ stand for the core domain causing selective apoptosis of cancer cells and the leucine zipper region, respectively. The VASA segment containing the D-I-N-N-N-N sequence is aligned with two Par-4 segments containing the E-L-N-N-N-L sequence and a Par-4 segment containing a N72A substitution. (C) ITC analysis. The Par-4 peptides were titrated into the indicated protein solutions, and the measured KD values are summarized in the table. The SSB-2 (HSVG) mutant contains the His116-Ser117-Val118-Gly119 sequence (see text). (D) (His)6-tag pull-down assay. Each of the wild-type and N72A mutant Par-4 (100 mg for each) was mixed with (His)6-tagged SSB-1 (50 mg) and Ni2+-chelating resin. After vigorous washing, the resin-bound proteins were analyzed by denaturing gel electrophoresis.

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overall sequence similarity with VASA but contains a stretch of Glu68-Leu69-Asn70-Asn71-Asn72-Leu73 (E-L-N-N-N-L) at the N-terminal domain of the protein (Figure 3B). Based on the existence of the N-N-N sequence motif, we prepared two peptides identical to residues 53–77 or 67–81 of Par-4 and a mutant Par-4 peptide (residues 67–81) containing a N72A substitution (Figure 3B). The interactions of the peptides with recombinant SSB-1 in complex with Elongin BC were analyzed by ITC. While the two wild-type peptides, both containing the E-L-N-N-N-L sequence, interacted with the SSB-1-Elongin BC complex with a KD of 146 nM and 180 nM, respectively (Figure 3C), the mutant peptide exhibited a poor binding affinity which could not be quantified by this method. We also produced full-length Par-4 and a mutant Par-4 protein containing the N72A substitution. The interactions of the two proteins with the SSB-1-Elongin BC complex were analyzed by (His)6-tag pull-down assay. As expected, the wild-type Par-4 interacted strongly with the SSB-1-Elongin BC complex, and the N72A mutation largely abrogated this interaction (Figure 3D), indicating that the structure- and sequence-based prediction of the recognition sequence crucial for the interaction between the two proteins was largely correct. Next, we titrated the Par-4 peptide (residues 53–77) into the wild-type and W221L mutant GUSTAVUS, both in complex with Elongin BC. As shown in Figure 3C, the binding affinity between the Par-4 peptide and the wild-type GUSTAVUS was estimated to be 174 nM, which is nearly equivalent to the binding affinity between the peptide and SSB-1. In contrast, the peptide exhibited extremely decreased binding affinity for the mutated protein (KD of 12 mM). These data indicate that the pocket on surface A of SSB-1 is indistinguishable from that of GUSTAVUS, and its interaction with the N-N-N motif is the prime contributor to the observed binding affinity. Compared with GUSTAVUS or SSB-1, SSB-2 interacted with the Par-4 peptide with much weaker affinity (Figure 3C; KD of 4.7 mM). The lower affinity appeared to arise from the sequence difference at the b7 region: Gln116-Thr117-Asp118-His119 in place of His-SerVal-Gly in SSB-1, SSB-4, and GUSTAVUS (Figure 3A). Modeling the four residues of SSB-2 on the structure of GUSTAVUS showed that the backbone torsion angles of His119 in SSB-2 should be different from those of the corresponding residue Gly132 in GUSTAVUS in order to have an energetically allowed main-chain conformation. Consequently, Tyr120, next to His119 in SSB-2, would have main- and side-chain conformations different from those of Tyr133 in GUSTAVUS, which is a critical residue in the interaction with the N-N-N motif of peptide I (Figure 2A). A slight displacement of Tyr120 in SSB-2 compared with the position of Tyr133 in GUSTAVUS would adversely affect the interaction of SSB-2 with the Par-4 peptide. To confirm this prediction, we prepared a mutant protein of SSB-2 in which all the four residues at the b7 region were substituted to the corresponding residues (His-Ser-Val-Gly) of GUSTAVUS, and the interaction of the mutated SSB-2 protein with the Par-4 peptide (residues 53–77) was analyzed by ITC. The mutated SSB-2 protein interacted with the peptide with w4-fold increased affinity (Figure 3C; KD of 1.2 mM) compared with the wild-type protein.

Table 2. D/E/N-L/M-N-N-N-X Motifs in the Drosophila Proteins Interacting with GUSTAVUS in Yeast Two-Hybrid Screening Protein (GenBank Accession Code) a

CG7134-PA (NP_609153) CG5376 (NP_651054)b CG13631 (NP_648509)b CG10348 (NP_609904)b cup/CG11181-PA (NP_523493)b a b

Putative Target Sequence 627 N-L-N-N-N-N 632 13 D-L-N-N-N-I 18 10 D-L-N-N-N-Y 15 125 E-L-N-N-N-H 130 407 N-M-N-N-N-N 412

Stanyon et al., 2004. Giot et al., 2003.

Because the mutation increased the affinity of SSB-2 for the Par-4 peptide, but not to a level similar to that of GUSTAVUS or SSB-1, these data indicate that the sequence difference at the b7 region of SSB-2 is partly responsible for the lower binding affinity of SSB-2 for Par-4. SSB and F Box-SPRY Proteins May Regulate Multiple Target Proteins The recognition of the six-residue sequence (D/E-I/L-NN-N-X) by the B30.2/SPRY domain of GUSTAVUS as the crucial motif suggests that the protein would bind a number of Drosophila proteins. This is because an identical segment can be found statistically in 1 out of 4000 theoretical proteins composed of 200 residues. In fact, five proteins identified as GUSTAVUS interactors by high throughput yeast two-hybrid screenings (Giot et al., 2003; Stanyon et al., 2004) contain a six-residue sequence identical to D/E/N-L/M-N-N-N-X (Table 2), strongly suggesting that these proteins are all cellular target proteins recognized by GUSTAVUS. As described above, the B30.2/SPRY domain of GUSTAVUS exhibits high sequence homology with those of three human SSB family proteins. Similarly, the B30.2/SPRY domain of GUSTAVUS shares 38%–40% sequence identity with the F box-SPRY protein orthologs, including mammalian FBXO45 proteins and C. elegans F box synaptic protein 1 (FSN-1) (Figure S2A). Furthermore, the pocketforming residues in GUSTAVUS are identically conserved in the F box-SPRY proteins, except for Val84 and Trp221, which are homologously substituted with isoleucine and tyrosine, respectively (Figure S2A). Mapping of the conserved residues onto the structure of GUSTAVUS shows that surface A is decorated with many of these residues (Figure S2B). The finding suggests that a nearly identical or similar pocket should be present in these SSB and F box-SPRY proteins and that it would recognize the D-I-N-N-N-X or a similar sequence motif present in multiple target proteins. A sequence analysis of 40,876 human proteins revealed that 30 cytoplasmic proteins, including Par-4, contain the D/N/E/Q-I/L/M-N-N-N-X motif in a predicted random coil region (data not shown). Although in-depth analyses (e.g., cell-based protein-protein interaction) are required, at least several of the identified proteins are likely to be the cellular targets of SSB-1 and SSB-4. Usually, the recognition subunit of a cullin-based E3 ubiquitin ligase binds several target proteins to regulate distinct processes in response to different signals (Petroski and Deshaies, 2005). For example, SOCS-1, which contains a central SH2 domain and a C-terminal

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SOCS box, interacts with more than 18 different proteins via the SH2 domain and regulates the activity of the interacting proteins (Ilangumaran et al., 2004). FBXW1/ b-TrCP1, which is composed of an F box and a WD40 domain, targets more than ten different substrate proteins (Nakayama and Nakayama, 2005), presumably via the WD40 domain that recognizes a six-residue sequence motif containing two phosphorylated serine residues (Wu et al., 2003). Likewise, using the B30.2/ SPRY domain, the SSB family and F box-SPRY proteins may target multiple proteins for regulation by ubiquitination. Presence of a Preformed Pocket on Surface A in Other B30.2/SPRY Domains In order to find whether the presence of a pocket on the conformationally rigid protein-binding site observed for GUSTAVUS may be a common feature in other B30.2/ SPRY domains, we first examined the structure of the PRY-SPRY domain of the 19q13.4.1-encoded protein (hereafter referred to as PSD), the only other related high-resolution structure available to date. The crystals of PSD contained four protein molecules in the asymmetric unit, which allows four independent views of the protein structure. A structural superposition of the four molecules revealed that the six loops (loop A to loop F) on the surface corresponding to surface A of GUSTAVUS exhibit remarkably similar conformations between the four molecules (Figure 4A): the rmsd values for the main-chain atoms of these loops (56 residues) were less than 1.02 A˚ for any compared pair of the four molecules. The conformational rigidity of these loops also arises from the intricate intramolecular interactions holding the loops in nearly fixed positions (data not shown). Remarkably, a structural superposition of PSD on GUSTAVUS shows that surface A of PSD has a prominent pocket, whose location is spatially similar to that of the pocket in GUSTAVUS (Figure 4A). Next, we analyzed the primary sequences of the TRIM family proteins that constitute the largest group of the B30.2/SPRY domain-containing proteins. The B30.2/ SPRY domains of the TRIM family proteins contain four variable regions (v1 to v4), in which sequence variations are greater than in the other regions of the domains (Song et al., 2005a). The variable regions v1, v2, v3, and v4 include loops B, D, F, and E, respectively, and exhibit substantial length variations as well as extensive amino acid differences (Figure S3). However, a group of 17 human TRIM family proteins exhibits variable region lengths in a much narrower range (19–21, 11–13, and 20–23 residues for v1, v2, and v3 regions, respectively), which is defined as the consensus lengths of the variable regions (Song et al., 2005a). By a multiple sequence alignment, we noted that PSD also has the same consensus lengths (20, 12, and 23 residues for v1, v2, and v3) and exhibits high sequence similarity (51% on average) with the B30.2/SPRY domains of the 17 TRIM proteins (Figure 4B). Furthermore, the three variable regions in PSD also exhibit high sequence similarity (49% on average) with the corresponding regions in the 17 TRIM proteins, reaching 52% and 58% with Ro52 and Pyrin, respectively. According to the sequence alignment, loop A to loop F of PSD contains 18 out of 26 residues conserved in the corresponding loops

of the 17 TRIM proteins. These residues are mostly buried and extensively involved in the interactions between loops A and B, loops B and D, or loop D and a b sheet (Figure 4C), indicating that not only the core structure but also the conformations of these loops in the 17 TRIM proteins should be closely similar to those of PSD. Remarkably, two hot spot mutations (M680I and M694V) in Pyrin causing FMF (Bakkaloglu, 2003) map exactly on the wall of the pocket of PSD, and four other FMFcausing mutations map on or right near the rim of this pocket (Figures 4A and 4B). These observations strongly suggest that the B30.2/SPRY domains of the 17 human TRIM family proteins also have a preformed pocket on surface A, which would play a primary role in the recognition of a short but specific sequence motif. Perspective In the mechanism by which the VASA peptide is recognized by the B30.2/SPRY domain of GUSTAVUS, two features are overwhelming. First, the B30.2/SPRY domain binds the linear sequence motif rather than a conformational motif (i.e., different regions of the target). Second, the target binding surface is conformationally rigid, and the preformed pocket interacting with the short sequence motif accounts for the binding affinity. These observations provide a rational ground to search for multiple target proteins containing a common sequence motif recognized by each of the SSB and F box-SPRY proteins. Since the two delineated structural features are presumed to be common in PSD and the 17 TRIM family proteins, peptide library screening appears to be the method of choice for identifying critical sequence motifs recognized by these and possibly other B30.2/SPRY domain-containing proteins. Such an experimental approach, phage display-driven selection of short recognition sequences, led to the identification of interaction partners of 28 yeast SH3 domains (Tong et al., 2002) and the GYF domain of a cellular adaptor protein CD2BP2 (Kofler et al., 2005). Finally, the presented information of the protein-protein interaction between GUSTAVUS and VASA represents the highest level attained for any B30.2/SPRY domain-containing proteins and sets the stage for the investigation of the physiological consequences of a specific disruption of the interaction between VASA and GUSTAVUS via a transgenic approach using site-directed mutants of the two proteins. Similar studies can be initiated for investigating the physiological significance of the Par-4 interaction with the SSB proteins. Experimental Procedures Preparation of GUSTAVUS in Complex with the VASA Peptide for Crystallization The Drosophila GUSTAVUS gene coding for residues 29–234 was amplified by polymerase chain reaction and ligated into the pPROEX vector (Invitrogen). The vector was transformed in Escherichia coli BL21 (DE3) RIG strain (Novagen). The protein was expressed at 15 C overnight and purified using a Ni-NTA column (QIAGEN) and a HiTrap Q anion exchange column (Amersham Biosciences). The N-terminal (His)6 tag attached to GUSTAVUS was removed by TEV protease, and the protein was further purified with Ni-NTA and HiTrap Q columns. The final protein sample was mixed with a 2-fold molar excess of the synthetic peptide (Peptron) identical to residues 184–203 of VASA. The mixture was loaded on a Superdex 75 gel filtration column (Amersham Biosciences), and the fraction

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Figure 4. Presence of a Preformed Pocket in PSD and a Putative Pocket in Other B30.2/SPRY Domains (A) A preformed pocket on surface A of PSD. The ribbon drawing and surface representation of PSD (PDB code 2FBE) are in the same orientation with GUSTAVUS. The presence of a pocket in PSD (middle) and GUSTAVUS (right) is highlighted by coloring the rim residues. The red meshes on

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containing the complex was concentrated to 12 mg/ml in 20 mM HEPES buffer (pH 7.5) and used for crystallization. Preparation of VASA, Par-4, and the SSB Family Proteins The coding sequence for Drosophila VASA was ligated into the pPROEX. The vector was transformed into E. coli BL21 (DE3) RIG strain. The expressed protein was purified using Ni-NTA and HiTrap Q columns. After removal of the N-terminal (His)6 tag by TEV protease, the protein was further purified using Ni-NTA and HiTrap Q columns. A standard protocol was used to generate the VASA mutant gene, and the mutant protein was prepared according to the procedure used for the purification of wild-type VASA. Human Par-4 was produced as an N-terminal GST fusion protein in E. coli BL21 (DE3) RIG strain and purified using glutathioneagarose resin (Peptron). After removal of the N-terminal GST tag by TEV protease, the protein was further purified using a HiTrap Q column. The N72A mutant Par-4 protein was prepared according to the procedure used for the purification of the wild-type protein. The coding sequence for human SSB-1 was ligated into the pPROEX vector. Each of the coding sequences for mouse Elongin B (full length) and Elongin C (residues 17–112) was cloned into pET30a (Novagen). From these vectors, a three-promoter expression vector was constructed, in which SSB-1, Elongin C, and Elongin B genes are sequentially arranged and controlled by Trc, T7, and T7 promoter, respectively. The three-promoter vector was transformed into E. coli BL21 (DE3) RIG strain. The expressed protein complex was purified using Ni-NTA and HiTrap Q columns. After removal of the N-terminal (His)6 tag by TEV protease, the protein complex was further purified using Ni-NTA and HiTrap Q columns. SSB-2 in complex with Elongin BC was produced and purified according to the procedures used for SSB-1. The wild-type and W221L mutant GUSTAVUS (residues 29–253) in complex with Elongin BC were produced as reported (Woo et al., 2006). Crystallization and Structure Determination Crystals of GUSTAVUS in complex with peptide I were obtained by the hanging-drop vapor-diffusion method at 20 C by mixing and equilibrating 1 ml each of the protein solution and a precipitant solution containing 10% (w/v) PEG 8000, 0.2 M calcium acetate, and 0.1 M imidazole (pH 7.0). The crystals belonged to the space group P41212 and contained two complexes in the asymmetric unit. A diffraction data set at 2.2 A˚ was collected on the beam line NW12A at Photon Factory in Japan and processed using the programs DENZO and SCALEPACK (Otwinowski and Minor, 1997). The structure was determined by the molecular replacement method with the CCP4 version of MOLREP (Vagin and Teplyakov, 1997) using the structure of GUSTAVUS (residues 35–234, PDB code 2FNJ) as a search model. The final model does not include residues 29–35 and 170–174 of GUSTAVUS, whose electron densities were not observed or were very weak. Crystallographic data statistics are summarized in Table 1. Isothermal Titration Calorimetry All measurements were carried out at 26 C on a MicroCalorimetry System (MicroCal). SSB-1, SSB-2, GUSTAVUS, and the W221L mutant GUSTAVUS in complex with Elongin BC were dialyzed against 10 mM phosphate-buffered saline solution (pH 7.4). The chemically synthesized VASA and Par-4 peptides (Peptron) were dissolved in the same buffer. The samples were degassed for 20 min and centrifuged to remove any residuals prior to the measurements. The experiments were carried out by titrating 0.15–0.5 mM peptides into 5–10 mM protein. Dilution enthalpies were determined in separate experiments (titrant into buffer) and subtracted from the enthalpies of the binding between the protein and the titrant. Data were

analyzed using the Origin software (OriginLab). The KD values were deduced from curve fittings of the integrated heat per mol of added titrants. Accession Numbers for the Protein Sequences The accession numbers in the sequence databases are GUSTAVUS (NP_724402.1), SSB-1 (NP_079382.2), SSB-2 (NP_116030.1), SSB-3 (NP_543137.2), SSB-4 (NP_543138.1), FBXO45 (NP_775615.2), FSN-1 (NP_498046.1), CG4643-PA (AAF58404.1), Pyrin (NP_000234.1), Ro52 (AAH10861.1), GNIP (NP_976038), TRIM10 (AAG53495.1), TRIM11 (NP_660215.1), TRIM15 (NP_150232.1), TRIM25 (AAH16924.1), TRIM26 (AAH32297.1), TRIM27 (AAG50172.1), TRIM38 (AAH26930.1), TRIM39 (AAH07661.1), TRIM50 (NP_835226.1), TRIM58 (NP_056246.2), TRIM60 (AAI00987.1), TRIM62 (CAH70402.1), TRIM68 (NP_060543.5), TRIM69 (ABB18376.1), TRIM4 (NP_148977.1), TRIM5a (NP_149023.1), MID1 (NP_150632.1), and TRIM36 (NP_061170.2). Supplemental Data Supplemental Data include three figures and can be found with this article online at http://www.molecule.org/cgi/content/full/24/6/967/ DC1/. Acknowledgments We thank Dr. S. Kim for the help with the analysis of human protein sequences. This study made use of the beam line NW12A at Photon Factory in Japan and was supported by Creative Research Initiatives (Center for Biomolecular Recognition) of MOST/KOSEF of Korea. J.-S.W. and H.-Y.S. were supported by the Brain Korea 21 Project. Received: July 31, 2006 Revised: October 1, 2006 Accepted: November 9, 2006 Published: December 28, 2006 References Bakkaloglu, A. (2003). Familial Mediterranean fever. Pediatr. Nephrol. 18, 853–859. El-Guendy, N., and Rangnekar, V.M. (2003). Apoptosis by Par-4 in cancer and neurodegenerative diseases. Exp. Cell Res. 283, 51–66. Giot, L., Bader, J.S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao, Y.L., Ooi, C.E., Godwin, B., Vitols, E., et al. (2003). A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736. Grutter, C., Briand, C., Capitani, G., Mittl, P.R., Papin, S., Tschopp, J., and Grutter, M.G. (2006). Structure of the PRYSPRY-domain: implications for autoinflammatory diseases. FEBS Lett. 580, 99–106. Gurumurthy, S., and Rangnekar, V.M. (2004). Par-4 inducible apoptosis in prostate cancer cells. J. Cell. Biochem. 91, 504–512. Ilangumaran, S., Ramanathan, S., and Rottapel, R. (2004). Regulation of the immune system by SOCS family adaptor proteins. Semin. Immunol. 16, 351–365. Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R.C., Conaway, J.W., and Nakayama, K.I. (2004). VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev. 18, 3055– 3065. Kile, B.T., Schulman, B.A., Alexander, W.S., Nicola, N.A., Martin, H.M., and Hilton, D.J. (2002). The SOCS box: a tale of destruction and degradation. Trends Biochem. Sci. 27, 235–241.

PSD indicate the mapping of the FMF-causing mutations in Pyrin (red letters). The corresponding residues of PSD are in parentheses. ‘‘del’’ stands for deletional mutation. (B) High sequence homology between PSD and 17 TRIM family proteins. Aligned are the sequences of the B30.2/SPRY domains of PSD and 17 TRIM family proteins (see text). The variable regions are indicated at the bottom of the alignment. Cyan and pink columns indicate the residues similarly conserved in more than 14 and 10 of the aligned proteins, respectively. Magenta and red boxes indicate the pocket-forming residues of PSD and the residues of Pyrin mutated in the FMF patients, respectively. (C) Location of the conserved residues. On the ribbon drawing of the PSD structure, the conserved residues are shown as sticks in the same color as in (B). The circle indicates the location of the pocket on surface A.

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Accession Numbers The coordinates of the GUSTAVUS-peptide I structure have been deposited in the Protein Data Bank with the accession code of 2IHS.