Structural Basis for Specific Binding of the Gads SH3 Domain to an RxxK Motif-Containing SLP-76 Peptide

Molecular Cell, Vol. 11, 471–481, February, 2003, Copyright 2003 by Cell Press

Structural Basis for Specific Binding of the Gads SH3 Domain to an RxxK Motif-Containing SLP-76 Peptide: A Novel Mode of Peptide Recognition Qin Liu,1 Donna Berry,2,7 Piers Nash,3,7 Tony Pawson,3,4 C. Jane McGlade,2,5 and Shawn Shun-Cheng Li1,6,* 1 Department of Biochemistry Faculty of Medicine and Dentistry University of Western Ontario London, Ontario N6A 5C1 2 The Arthur and Sonia Labatt Brain Tumor Research Center Hospital for Sick Children Toronto M5G 1X8 3 Samuel Lunenfeld Research Institute Mount Sinai Hospital Toronto M5G 1X5 4 Department of Medical Genetics and Microbiology 5 Department of Medical Biophysics University of Toronto 1 Kings College Circle Toronto M5S 1A8 6 Child Health Research Institute 800 Commissioners Road East London, Ontario N6C 2V5 Canada

Summary The SH3 domain, which normally recognizes prolinerich sequences, has the potential to bind motifs with an RxxK consensus. To explore this novel specificity, we have determined the solution structure of the Gads T cell adaptor C-terminal SH3 domain in complex with an RSTK-containing peptide, representing its physiological binding site on the SLP-76 docking protein. The SLP-76 peptide engages four distinct binding pockets on the surface of the Gads SH3 domain and upon binding adopts a unique structure characterized by a right-handed 310 helix at the RSTK locus, in contrast to the left-handed polyproline type II helix formed by canonical proline-rich SH3 ligands. The structure, and supporting mutagenesis and peptide binding data, reveal a novel mode of ligand recognition by SH3 domains. Introduction The SH3 domain is one of the most abundant protein interaction modules found in nature (Mayer 2001; Tong et al., 2002). It is involved in a wide range of cellular functions including signal transduction, enzymatic regulation, membrane transport, cytoskeletal rearrangement, and organelle biogenesis (Mayer 2001). SH3 domains are approximately 60 residues in length and share significant sequence identity, as well as a common structure featuring two antiparallel ␤ sheets packed against each other (Yu et al., 1992, 1994; Musacchio et al., 1992; Noble et al., 1993; Lim and Richards, 1994; Feng et al., 1994, 1995; Lee et al., 1996). The majority of SH3 do*Correspondence: [email protected] 7 These authors contributed equally to this work.

mains characterized to date bind to proline-rich sequences containing a core element, PxxP, through a set of conserved surface residues (Lim and Richards, 1994; Feng et al., 1995; Mayer 2001). The bound peptide invariably assumes a left-handed polyproline type II (PPII) helical conformation, and the prolines in the peptide core directly contact the SH3 domain through the two X-Pro dipeptide units (where X is usually a hydrophobic residue) (Feng et al., 1994). Due to the pseudosymmetrical nature of the PPII helix, peptide ligands potentially bind a given SH3 domain in either one of two opposite directions governed by the location of a positively charged residue, usually Arg, which often precedes or follows the PxxP core element (Lim et al., 1994; Feng et al., 1994, 1995). Thus, class I ligands with the consensus sequence RxXPxXP bind to SH3 domains in a direction opposite to that for class II ligands possessing a generic sequence of XPxXPxR (where “x” denotes any amino acid while “X” is usually a hydrophobic residue). Additional contacts outside of the core can confer enhanced specificity and binding affinity. For instance, high-affinity binding of the tyrosine phosphatase PEP to the C-terminal Src kinase (Csk) SH3 domain involves not only a conventional XPxXPxR-containing motif in PEP, but also two hydrophobic residues (Ile and Val) C-terminal to this motif (Ghose et al., 2001). Similarly, the SH3 domain of p67phox binds a proline-rich region (PRR) at the C-terminal tail of p47phox but also makes additional contacts with a stretch of 20 amino acids immediately following the PRR, resulting in a 1000-fold increase in affinity (Kami et al., 2002). Some SH3 domains, however, recognize peptide sequences devoid of the core PxxP element, suggesting that they possess novel binding modes. In accord with this notion, the SH3 domains from the tyrosine kinase substrate Eps8 and related proteins bind selectively to sequences containing a PxxDY motif (Mongiovi et al., 1999). Recent data have also suggested that some SH3 domains may bind to peptide motifs dominated by basic residues. For example, the SH3 domains of Fyn and Fyn binding protein, Fyb/SLAP130, engage a site in the immune cell adaptor SKAP55 with a consensus sequence RKxxYxxY that lacks a proline (Kang et al., 2000). The SH3 domains of the STAM family of proteins, including STAM, EAST, and Hbp, which are involved in cytokinemediated signaling and receptor-mediated endocytosis and exocytosis (Lohi and Lehto, 2001), bind to a consensus motif Px(V/I)(D/N)RxxKP conserved in AMSH and UBPY (Kato et al., 2000). Interestingly, similar motifs have also been identified in several other proteins, including SLP-76 and the scaffolding proteins Gab1/ Gab2, which have been shown to mediate specific interactions with the C-terminal SH3 domains of the SH2/ SH3 adaptors Gads and Grb2, respectively (Lock et al., 2000; Lewitzky et al., 2001; Berry et al., 2002). The specificity of these SH3 domain-ligand interactions appears to be determined largely by the novel RxxK core element, since both R and K in the consensus are indispensable for binding (Berry et al., 2002). This ability to recognize an RxxK core element is likely an evolution-

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Figure 1. Solution Structure of a Gads SH3-C Domain – SLP-76 Peptide Complex (A) Stereo view of a superposition of 20 lowest-energy structures of the Gads SH3-C domain – SLP-76 peptide complex in backbone traces. The Gads SH3-C domain (residues 265–321) is shown in green and the SLP-76 peptide in violet. The peptide has the sequence A1PSIDRSTKPA11, of which the first ten residues were taken from the Gads SH3-C – binding site in SLP-76, and the last Ala was added to reduce end effects. Ala11 does not interact with the SH3 domain (see Figure 3B) and is hence omitted from all figures for clarity. (B) Ribbon representation of the same complex generated using coordinates of the lowest-energy structure. The ␤ strands and the RT- and n-Src loops of the SH3 domain are labeled in black. The peptide backbone is depicted in violet with side chains shown in pale green or dark green (for residues located at the peptide-protein interface).

arily conserved property, since the C-terminal SH3 domain of the Drosophila homolog of Grb2, Drk, associates with Dos via two RxxK-containing sites, thereby coupling Dos to the Sevenless (Sev) receptor tyrosine kinase (Feller et al., 2002). Binding of Gads to SLP-76 serves an important physiological role in coupling the latter to membrane-associated LAT (Linker for Activated T cells) upon T cell receptor (TCR) activation (reviewed in Liu et al., 2001). In thymocytes derived from Gads-deficient mice, signaling through the TCR-associated molecule CD3 is impaired, and SLP-76 is largely uncoupled from LAT in these cells (Yoder et al., 2001). SLP-76 bears hallmarks of docking proteins—it has an N-terminal acidic region containing several Tyr residues within consensus motifs for SH2 binding, which in their phosphorylated state bind the Vav SH2 domain (Tuosto et al., 1996), an extended proline-rich region that binds to the SH3 domains of PLC␥1 (Yablonski et al., 2001) and Gads (Liu et al., 1999; Law et al., 1999); and a C-terminal SH2 domain that interacts with FYB/SLAP-130 (ADAP) (da Silva et al., 1997; Musci et al., 1997; Griffiths et al., 2001). While the PLC-␥1 SH3 domain may interact with SLP-76 through one or more proline-rich motifs (Yablonski et al., 2001), the Gads SH3-C domain binds in a highly selective manner to a short segment in SLP-76 that spans residues 233–242 and lacks a conventional PxxP motif (Berry et

al., 2002). A peptide derived from this binding site and comprised of the sequence APSIDRSTKPA binds to the Gads SH3-C domain with a dissociation constant of 240 nM in vitro, which ranks among the strongest interactions observed between an SH3 domain and a naturally occurring ligand (Berry et al., 2002). The Grb2 SH3-C domain also binds this motif in vitro, but with 40-fold lower affinity, such that in T cells the physiological interaction selectively involves Gads and SLP-76 (Liu et al., 1999; Berry et al., 2002). While the structural basis for the binding of SH3 domains to PxxP motifs has been extensively explored (reviewed in Mayer, 2001), there are no data to explain how SH3 domains might recognize the entirely distinct, and biologically important, RxxK elements. To address this issue, we have solved the structure of the Gads SH3-C domain in complex with the peptide corresponding to its binding site in SLP-76. This has revealed a novel mode of SH3-peptide recognition, which is supported by mutagenesis and peptide binding studies. Results The SLP-76 Peptide Engages Four Distinctive Binding Pockets on the Gads SH3-C Domain The structure of the Gads SH3-C domain in complex with the SLP-76 peptide (APSIDRSTKPA) was determined by

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Table 1. Statistics for the Final 20 Structures of the Gads SH3-C Domain – SLP-76 Peptide Complex Rmsd from experimental distance restraints 1973 Unambiguous (A˚) 189 Ambiguous (A˚) 44 Hydrogen bond (A˚)a 70 Dihedral angle (⬚) Rmsd versus average structure (A˚)b Backbone atoms All heavy atoms Deviation from idealized geometry Bond (A˚) Angle (⬚) Improper (⬚) Non-bonded energies in PROLSQ force field (kcal.mol⫺1) Van der Waals Electrostatic Residues in allowed φ/␺ regions of the Ramachandran plot (%) Most favored regions Additionally allowed regions Generously allowed regions Disallowed regions a b

0.014 ⫾ 0.002 0.013 ⫾ 0.004 0.010 ⫾ 0.002 1.44 ⫾ 0.03 0.43 ⫾ 0.08 1.09 ⫾ 0.13 0.0021 ⫾ 0.0001 0.401 ⫾ 0.007 0.270 ⫾ 0.009 ⫺131 ⫾ 30 ⫺236 ⫾ 8 75.7 21.5 1.1 1.7

Two restraints were used to derive each hydrogen bond. The values shown represent residues from Val265 to Arg322 of the Gads SH3-C domain and from Ala1 to Pro10 of the SLP-76 peptide.

nuclear magnetic resonance (NMR) spectroscopy. An overlay of an ensemble of 20 structures of lowest average energy is displayed in Figure 1A, with the statistics of these structures given in Table 1. Except for a few residues at the extreme N- and C termini of the protein, both the SH3 domain and the bound peptide are well defined in the complex (Table 1). The Gads SH3-C domain adopts a typical SH3 fold consisting of two antiparallel ␤ sheets formed by five ␤ strands, with a single turn of 310 helix between strands ␤4 and ␤5 (Figure 1B). The structure of the peptide, however, is completely different from those of other SH3 ligands reported to date. The most notable feature is that the RSTK moiety of the peptide assumes a right-handed 310 helical conformation instead of the conventional left-handed PPII helix formed by proline-rich sequences. The 310 helix is preceded by an extended N terminus that includes residues Ala1 to Asp5 (numbered from the N- to C terminus, Figure 1B). The overall shape of the peptide resembles an inverted letter V, with Arg6 located at the tip. As with complexes between SH3 domains and PxxP class ligands, the peptide binding surface of the Gads SH3-C is located in the area bordered by strands ␤4 and ␤5, the C-terminal 310 helix, the RT loop that connects strands ␤1 and ␤2, and the n-Src loop that joins strands ␤2 and ␤3 (Figure 1B). Critical residues in the Gads SH3-C domain involved in peptide recognition include Tyr272, Glu275, Leu277, Glu278, Glu281, Trp300, Leu311, Pro313, and Tyr316, and the locations of these residues in the complex are shown in Figure 1B. All residues in the peptide displayed intermolecular NOEs, with residues Ala1, Pro2, Ile4, Arg6, Lys9, and Pro10 exhibiting extensive contacts with the SH3 domain. The orientation of the SLP-76 peptide with respect to the Gads SH3-C domain is akin to that of a class II peptide bound to the c-Src SH3 domain (Feng et al., 1994, 1995). As in the binding of the c-Src SH3 domain to a PxxP peptide (Feng et al., 1995), both hydrophobic and electrostatic interactions contribute to the formation of the Gads SH3-C – SLP-76 peptide complex. However, their

relative contributions to the total binding energy appear different in the two cases. Uniquely, the Gads SH3-C domain employs four binding pockets on the protein surface for recognition of the core peptide sequence, instead of the three used by c-Src SH3 and other conventional SH3 domains (Figure 2). These include two hydrophobic pockets on Gads SH3-C that accommodate the N-terminal residues of the peptide, a large, negatively charged surface that interacts with the RSTK moiety, and a small and relatively less well defined pocket for the C-terminal residue Pro10 (Figures 1B and 2A). The first hydrophobic pocket, formed by residues Tyr272 and Tyr316 (Figure 1B), is structurally equivalent to the pocket on the c-Src SH3 domain used to bind the first XP dipeptide unit in the consensus sequence XPxXPxR of the class II ligands (e.g., peptide APP12 in Figure 2B). This pocket is occupied by the dipeptide unit Ala1-Pro2 in the Gads SH3-C complex. The second hydrophobic pocket formed by Tyr316, Pro313, and Trp300 efficiently accommodates the side chain of Ile4. The location of this pocket is similar to the second hydrophobic pocket in the c-Src SH3 domain, which forms a hydrophobic groove ideal for accommodating the second XP dipeptide (Leu4-Pro5 in peptide APP12, Figure 2B). In the Gads SH3-C domain, however, the corresponding hydrophobic groove is enclosed on one side by the side chain of Glu275, creating a pocket that is smaller but deeper than that in the c-Src SH3 domain (Figure 2A). While the side chain of Ile4 fits nicely and protrudes deeply into the pocket, this Gads SH3-C pocket is too small to accommodate an XP unit (Figures 2A and 2B). This likely explains why the Gads SH3-C domain does not bind with high affinity to PxxP-containing peptides and why substitution of Ile4 in the SLP76 peptide by a hydrophilic residue abolishes its binding to Gads C-SH3 domain (see below). The third pocket of the Gads SH3-C domain is formed by residues Glu278 and Glu281 and is cushioned at the base by Trp300. This pocket mediates a network of extensive interactions with the side chains of Arg6 and

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Figure 2. A Comparison of Peptide Binding Surfaces between the Gads and c-Src SH3 Domains (A) Surface representation of the Gads SH3-C domain – SLP-76 peptide complex. Areas of positive and negative charges are shown in blue and red, respectively. Residues in the peptides that occupy the four binding pockets on the SH3 domain surface (identified in dotted circles) are labeled in black. Residue Glu275 of the protein, which encloses the second pocket, is labeled in red. (B) Surface representation of the c-Src SH3 domain – APP12 peptide complex (adapted from Feng et al., 1995). APP12 is a dodecapeptide that binds with high affinity to the c-Src SH3 domain (Kd ⫽ 1.2 ␮M). Since the last four residues of the peptide do not contribute significantly to SH3 binding (Feng et al., 1995), only the first eight residues of the peptide (A1PPLPPRN8 ) are shown for clarity. As in (A), key residues in peptide APP12 that engage the three binding pockets (identified as dotted circles) on the c-Src SH3 domain are labeled.

Lys9 via both salt bridges and hydrophobic contacts, as discussed in detail below. The fourth binding pocket on the Gads SH3-C domain is formed by residues Trp300 and Leu311, which anchor the C-terminal proline of the peptide (Figures 1B and 2A). A similar pocket is not found on the c-Src SH3 domain. It is worth noting that Trp300 is involved in the formation of the second, third, and fourth binding pockets. The pivotal role of Trp300 in nucleating the Gads SH3-C domain – SLP-76 complex is highlighted by the observation that its mutation to Ala completely eliminated Gads binding to SLP-76 (see below). Structure and Specificity of the RSTK Motif The most prominent feature of the SLP-76 peptide is the 310 helix formed by the critical RSTK residues (Figure 3A). The helical conformation orients the side chains of Arg6 and Lys9 in the same direction, enabling them to interact simultaneously, via salt bridges, with the acidic side chains of Glu278 and Glu281 in the RT loop, respectively. The side chain of Arg6 is slightly bent toward that of Lys9, making it capable of also interacting with Glu281 (Figure 3A). These charge-charge interactions are further strengthened by hydrophobic interactions between the acyl chains of Arg6 and Lys9 and the side chain of Trp300, which sits snugly at the base of the 310 helix. The side chain of Arg6 is also in close contact with that of Leu277, as evidenced by observable NOEs between the two residues. Other amino acids in the helix also display intermolecular NOEs; those between residues Thr8 of the peptide and Ser299 of the protein are particularly strong. Thus, the RSTK motif in the SLP-76 peptide participates in an array of interactions with the Gads

SH3-C domain through both electrostatic attractions and hydrophobic contacts. This is in contrast to the c-Src SH3 – APP12 peptide complex, where the only observable salt bridge occurs between residue Arg7 in the peptide and Asp99 of the protein (Feng et al., 1995; see also Figure 2). It is expected that the RSTK motif in the SLP-76 peptide would contribute significantly more energy than the single Arg7 in peptide APP12 in binding to their respective SH3 domains. How is the RSTK 310 helix formed in the peptide? Since stable helical conformations are rarely observed in structurally unrestrained short peptides, the formation of a single turn of helix at the RSTK locus is likely aided by factors both intrinsic and extrinsic to the peptide. Indeed, one notable stabilizing factor is found in residue Asp5, immediately preceding the helix. Asp is one of the best N-capping residues for ␣ helices (Doig and Baldwin, 1995), and Asp and Asn are also particularly favored for capping 310 helices since they can hydrogen bond to backbone NH groups while in the trans rotamer (Doig et al., 1997). Indeed, Asp5 displays strong NOEs to both the Ser7 side chain and Thr8 amide protons. The side chain carboxyl group of Asp5 is well positioned to form hydrogen bonds with the side chain of Ser7 or/ and the backbone amide of Thr8 so as to stabilize the RSTK helix (Figure 3A). These hydrogen-bonding interactions also induce a kink in the backbone between residues Asp5 and Arg6, resulting in the distinctive inverted “V” shape of the peptide (Figure 1B). Additional, but critical, stabilizing energy for the helix is perhaps derived from electrostatic interactions between the side chains of Arg6 and Lys9 and the acidic residues in the RT loop of the SH3 domain. Electrostatic interactions

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Figure 3. Structure and Interactions of the RSTK Motif-Containing SLP-76 Peptide (A) Conformation and interactions mediated by the RSTK site of the SLP-76 peptide (in stereo view). Backbone and side chain heavy (except N and O) atoms of the peptide are shown in cyan, while those of the SH3 domain are shown in gray. Nitrogen and oxygen atoms are depicted in blue and red, respectively. Residues are identified in cyan while interacting residues in the SH3 domain in black. Potential electrostatic and hydrogenbond interacting pairs are identified by arches. For simplicity, hydrogen atoms are not included in the figure. (B) An overlay of 15N-1H correlation spectra (HSQC) of peptide SLP-76 in free (colored in black) and bound (in red) states. The boxed peak is originated from the side chain of Arg6. Note that the intensity of the amide resonance of Ala11 is much greater than those of other residues and that its position moved very little from the free state, indicating that Ala11 is quite flexible in the bound state and hence unlikely involved in binding the SH3 domain.

between the peptide and the RT loop and the formation of a 310 helix in the RSTK motif are likely cooperative and mutually dependent events. While charge-charge interactions stabilize the helical conformation, the latter may in turn reinforce the former. Since the PPII helix is often formed in PxxP class ligands of SH3 domains prior to binding, we were interested to see whether the 310 helix observed in the SLP76 peptide was preformed. HSQC spectra recorded on a free 15N, 13C-labeled SLP-76 peptide sample produced a set of sharp peaks distributed in a narrow region along the 1H axis, suggesting that the free peptide is unstructured. Since more than one set of amide resonance peaks were observed in the HSQC, the free peptide likely undergoes structural transitions, perhaps mediated by the cis-trans isomerization of the Ala1-Pro2 or the Lys9Pro10 bond. Interestingly, these sharp peaks were replaced by a single set of nicely dispersed peaks when unlabeled Gads SH3-C domain was titrated into the peptide (Figure 3B), indicating that the helical structure of the peptide is induced upon binding to the SH3 domain rather than being preformed in solution.

Mutation of Key Residues in Both the SH3 Domain and the RxxK Peptide Affects Binding of Gads and SLP-76 In Vitro and In Vivo Six residues in the SLP-76 peptide, namely, Ala1, Pro2, Ile4, Arg6, Lys9, and Pro10, are located at the peptideprotein interface and contribute significantly to binding of the Gad SH3-C domain. To assess their relative contributions to binding, each of these residues (with the exception of Ala1) was replaced by an Ala, and the affinity of the corresponding peptides for the Gads SH3-C domain was measured in a competition assay using fluorescence polarization. We previously reported that substitution of either Arg6 or Lys9 by an Ala abrogated binding to the Gads SH3-C domain and that substitutions of either Pro2 or Pro10 resulted in significant loss of binding affinity for the corresponding peptides P2A and P10A (Berry et al., 2002; Figure 4A). As expected, replacement of both Pro residues by Ala in peptide P2A/P10A had a more dramatic effect on binding (Figure 4A). Substitution of Ile4 by either Ala (as in I4A) or Pro (as in I4P) also resulted in significant reduction in binding, pointing to the importance of maintaining the

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Figure 4. Specificity Determinants for the Interaction between the SLP-76 Peptide and the Gads SH3-C Domain (A) Peptide IC50 curves for the competition of binding to purified Gads SH3-C. FluoresceinSLP-76 wt peptide (Fl-APSIDRSTKPA) bound to Gads SH3-C was competed away by SLP76, SLP-76 mutant peptides, or a SOS peptide (VPPPVPPRRR) (Nash et al., 2002). Calculated IC50 values were obtained from the average of at least three independent experiments and are on the right of the competition curves. For purpose of comparison, relative IC50 values (with that of wt peptide set at 1.0) are given. (B) Equilibrium binding curves for Gads SH3-C mutants to fluorescein-SLP-76 peptide measured by fluorescence polarization. Binding data were analyzed using the Michaelis-Menton equation in order to obtain the dissociation constant (Kd) values. Average values from at least three independent experiments are reported.

side chain chemistry and hydrophobicity at this site. Interestingly, replacement of Asp5, a residue not directly involved in SH3 binding, by Ala led to a nearly 20-fold reduction in affinity (Figure 4A). This observation is in excellent agreement with the critical role for Asp5 in stabilizing the 310 helical conformation of the RSTK motif. To test the ability of the Gads SH3-C domain to bind PxxP-containing sequences, we replaced Asp5 with a Pro residue, thereby generating a peptide that contains a PSIP-motif. This peptide (D5P) was found to be capable of binding to the Gads SH3-C domain, although with over 10-fold lower affinity. A control PxxP class peptide derived from the SOS protein also bound weakly to the Gads SH3-C domain. These results demonstrate that PxxP-containing peptides are not favored ligands of the Gads SH3-C domain, possibly owing to the unique characteristics of its second hydrophobic binding pocket. To explore the stringency or flexibility of the RxxK motif, we synthesized peptides bearing R to K, and/or K to R substitutions, thus generating peptides containing a KxxK (peptide R6K), an RxxR (peptide K9R), or a KxxR (peptide R6K/K9R) motif. As shown in Figure 4A, none of these peptides was effective in competing for Gads SH3-C binding against the wild-type peptide. Of particular interest, the R/K swapping peptide, R6K/K9R, completely lost its affinity for Gads SH3-C. These results indicate that, unlike the PxxP motif, which can bind an SH3 domain in either one of two opposing directions (Lim et al., 1994; Feng et al., 1994, 1995), the interaction between the RxxK motif and the Gads SH3-C domain is unidirectional. Furthermore, the Gads SH3-C domain imposes stringent side chain requirements for the RxxK motif, since even conservative mutations such as those

seen in peptide R6K and K9R are not tolerated. This precision in ligand recognition by the Gads SH3-C domain may be rooted in its structure. Since Arg6 of the peptide interacts with both E278 and E281 of the Gads SH3-C domain via electrostatic interactions (Figure 3A), its replacement with a Lys residue would compromise the double salt bridges due to the single positive charge for the latter amino acid at physiological pH. In contrast, because Lys9 is in close proximity to E281 in the complex, an Arg residue in place of Lys9 would create steric hindrance with residue E281, due to the bulkiness of the Arg side chain (Figure 3A). Results from the peptide studies were confirmed in coprecipitation experiments using intact proteins expressed in Jurkat T cells. As shown in Figure 5A, SLP76 mutants P233A, P241A, I235A, and D236A (corresponding to peptides P1A, P10A, I4A, and D5A in Figure 4A) showed reduced binding to Gads. As with peptide binding studies, a D246 to Ala mutation had a more severe effect on binding than mutations involving Pro233, Pro241, or Ile235. Of note, mutant I235E showed no detectable binding to Gads, highlighting the critical role of hydrophobic interaction at position Ile4 of the SLP-76 peptide. The residues in the Gads SH3 domain involved in peptide recognition were also mutated singularly or in combination. Most single mutants, such as L277A, E278A, and E279A, had negligible or only moderate effects on binding either to the SLP-76 peptide in vitro (Figure 4B) or SLP-76 protein in T cells (Figure 5B), suggesting that the interaction between SLP-76 and Gads SH3-C is a cooperative event involving multiple residues and that none of the above residues alone plays a domi-

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Figure 5. Mutations of Residues at the Interface of the Complex Affect Binding of Gads to SLP-76 in Cells (A) Binding of Flag-tagged SLP-76 mutants to endogenous Gads protein in the SLP-76-deficient Jurkat cell line, J14. Alanine substitutions at P233, P241, I235, or D236A led to a reduction in coimmunoprecipation with Gads. Substitution of I235 with a proline residue also led to decreased binding, while a glutamate residue at position 235 completely abrogated binding. (B) Binding of Gads SH3-C domain mutants to SLP-76 in Jurkat T cells. Flag-tagged wt Gads protein was compared with Flag-tagged Gads mutants for ability to precipitate endogenous SLP-76. Compared to wt Gads, single Ala substitutions at L277 and E279 had mild effects on binding, while single substitutions at E278, E281, or W300, or double mutations at positions 277, 278, 279, and 281, greatly reduced or completely abolished binding.

nant role in binding. However, exceptions to this rule were also observed. Of particular note, mutation of residue E281 to an Ala (E281A) in Gads SH3-C abolished its interaction with SLP-76 in Jurkat T cells (Figure 5B) and markedly reduced its affinity for the SLP-76 peptide (Figure 4B). This observation is in excellent agreement with the structure of the Gads SH3-C –SLP-76 peptide complex, in which residue E281 plays a pivotal role in engaging the peptide through a double salt bridge with the RSTK motif. Similarly, replacement of Trp300, a residue involved in the formation of three of four binding pockets on the Gads SH3-C domain, by Ala led to a complete loss of binding to the SH3 domain both in vitro and in vivo (Figures 4B and 5B). It should be noted that this Trp residue is invariant in all SH3 domains identified to date. To test the collective effect of acidic residues of the RT loop in binding, we also generated double mutants such as LE-AA (277, 278), EE-AA (278, 279), and EE-AA (278, 281). The first two double mutants behaved very similarly to the single mutant E278A, while mutant EE-AA (278, 281) completely lost its affinity for SLP-76. Although the latter result was expected, given the effect of the single E281A substitution on SLP-76 association in cells, peptide binding data demonstrated that the effect of mutating both E278 and E281 was far more severe than mutating either residue alone (Figure 4B).

It is likely that the effect of mutating one acidic residue in the E278/E281 pair can be partly negated by neighboring acidic residues in the RT loop via conformational changes. However, mutating both E278 and E281 leads to a substantial loss of negative charges at the binding site for the RxxK motif, which cannot be simply compensated for by structural readjustments. Discussion The structure of the Gads SH3-C domain – SLP-76 peptide complex reveals a novel mode of peptide recognition which is distinct from those used by SH3 domains that recognize the PxxP motif. Apart from the presence of four, instead of three, binding sites on the Gads SH3-C domain, the SLP-76 peptide ligand assumes a unique structure that is characterized by a 310 helix at the RSTK locus and optimized for high-affinity binding. The structure also shows how an SH3 domain can select against an XP dipeptide unit, which is a hallmark of most SH3 domain-ligand interactions (Nguyen et al., 1998). All residues in the SLP-76 peptide, except Ala11, which is not a natural residue found in SLP-76, participate either in binding the SH3 domain or in maintaining the integrity of the peptide structure. These residues appear to be fine-tuned for binding the Gads SH3-C domain, since

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Figure 6. Conformational Change in the RT Loop Alters SH3 Domain Specificity Superposition of the Gads SH3-C – SLP-76 peptide complex (peptide in violet, protein in red) with the c-Src SH3 – peptide complex (peptide in gold, protein in green). Key residues in the two peptides are labeled, as are the RT- and n-Src loops.

substitution of any one amino acid in the peptide or SLP-76 invariably resulted in a loss of affinity for Gads SH3-C, albeit to varying degrees. In this regard, the RxxK motif can only bind the Gads SH3-C domain in one direction, distinct from the bi-directional interactions between PxxP motif-containing peptides and SH3 domains. In contrast to the stringent requirements for specific residues in the peptide, the Gads SH3-C domain itself is relatively resilient to mutations, likely due to the flexible nature of the RT loop. Our data suggest that there are both similarities and significant differences in the modes of ligand recognition between the Gads SH3-C domain and those SH3 domains that prefer PxxPcontaining peptides. Switching Specificity by “Loop Swing”—Comparison with the c-Src SH3 Domain – Peptide Complex Two questions were raised by our previous finding that the Gads SH3-C domain binds to an RxxK motif in SLP76: why does the Gads SH3-C domain preferentially recognize an RxxK to a PxxP motif, and why is this interaction so strong compared to conventional SH3 domain-peptide complexes? These questions can now be addressed by analysis of the structure of the GadsSLP-76 complex. Superimposing the Gads SH3-C domain over the c-Src SH3 domain reveals that the RT loops of the two domains differ significantly from each other (rmsd at 1.7 A˚ for the loops, compared with 0.9 A˚ for secondary structure elements, Figure 6). The base of the RT loop in the Gads SH3-C domain is shifted toward the n-Src loop while the tip of the loop swings away from the n-Src loop. This creates a binding groove that is narrower at the base and wider at the mouth than that in the c-Src SH3 domain (Figure 6). Movement of the RT loop also results in the closure of one side of the second hydrophobic binding groove by residue Glu275,

converting the binding groove into a pocket. The selective exclusion of an XP dipeptide unit from this pocket underlies the low affinity of the Gads SH3-C domain for PxxP-containing sequences. The swing of the tip of the RT loop away from the n-Src loop in the Gads SH3-C domain places the double acidic residues, Glu278 and Glu281, in perfect positions to interact with the basic side chains of the Arg and Lys in the RSTK 310 helix. These double charge-charge interactions play a much more prominent role in Gads SH3-C than the single salt bridge in the c-Src – peptide complex and contribute significantly to the total binding energy for the former complex. The unusually high affinity seen in binding of the Gads SH3-C to the SLP-76 peptide may also have its origin in the extensive hydrophobic contacts of the peptide ligand with the Gads SH3-C domain. As shown in Figure 6, the SLP-76 peptide as a whole sinks more deeply into the binding surface of Gads SH3-C than does the APP12 peptide in the APP12-c-Src SH3 complex. While interactions between the two XP units with the hydrophobic pockets of conventional SH3 domains are rather superficial, which is also why most SH3-peptide interactions are weak, the corresponding pockets in the Gads SH3-C domain engage the SLP-76 peptide more tightly (Figure 6). SH3 Domain-Ligand Interactions: With or Without Proline For conventional SH3 domain-PxxP motif interactions, Pro residues in the peptide serve dual roles—by providing a scaffold for the formation of the unique PPII helical structure and by interacting directly with the hydrophobic binding pockets on the SH3 domain. Although the PPII helix is a hallmark of SH3 domain binding ligands identified to date, proline is not absolutely required for its formation. For instance, the PPII structure has been observed in short peptides enriched in Ala (Shi et al., 2002), in peptides bound to the MHC-II molecules (Madden, 1995), and in SH3 binding sequences devoid of the PxxP motif (Xu et al., 1997). In the last instance, a PQGQ segment in the linker connecting the SH2 and the kinase domains of c-Src forms a PPII helix in complex with its own SH3 domain (Xu et al., 1997). However, this intramolecular interaction is unlikely of high affinity since the hydrophilic side chain of the last Gln in the PQGQ motif interacts unfavorably with the second hydrophobic pocket on the c-Src SH3 domain. Indeed, binding of the linker peptide to the SH3 domain is strengthened by concomitant interactions between the n-Src and RT loops of the SH3 domain and the kinase domain (Xu et al., 1997). In comparison, the only identifiable regular secondary structure in the SLP-76 peptide is a 310 helix formed at the RSTK locus. Although Pro residues in the SLP-76 peptide play significant roles in binding the Gads SH3C domain via hydrophobic interactions with the first and fourth pockets, they do not act as structural scaffolds as do those in PxxP motif-containing peptides. Residues N-terminal to the RSTK 310 helix in the SLP-76 peptide, including Pro2, are in a more elongated conformation than the PPII helix in the APP12 peptide, and there is only one intervening residue (Ser3) between Pro2 and Ile4 that interacts respectively with the first and second

Novel Mode of Peptide Binding by an SH3-C Domain 479

binding pockets, compared to two amino acids separating the Pro residues in the PxxP PPII helix (Figure 6). Our study shows that high-affinity binding of a peptide ligand to an SH3 domain is possible without the presence of a PPII helix. The structure of the Gads SH3 domain-SLP-76 peptide complex illustrates the highly adaptive nature of interaction domains. The peptide binding surfaces for such domains are often composed of residues originated from loops, that are both flexible in structure and variable among members of a given family of interaction domain. Variations in these regions can lead to modification of side chain chemistry at the protein-ligand interface and thereby provide a mechanism to generate diverse binding specificity, as also seen for other protein modules such as the SH2 and PTB domains (Li et al., 1998, 1999; Zwahlen et al., 2000; Hwang et al., 2002). The major specificity determinant in Gads SH3-C domain is the RT loop, in particular, the acidic residues within the loop. Thus, by modifying the side chain chemistry and/ or the location of interface residues, novel specificity can be generated. The unique mode of peptide recognition by the Gads SH3-C domain may be shared by other members of the SH3 family. Sequence alignment suggests that the SH3 domains of Hbp and STAM (Kato et al., 2000; Lohi and Lehto, 2001) display high sequence identity in their RT loops to those in the Gads and Grb2 SH3-C domain and may therefore employ a similar mode of peptide recognition as the latter. However, subtle differences could exist among these SH3 domains despite the high level of sequence conservation, as indeed is observed between the Gads and Grb2 SH3-C domains (Berry et al., 2002). The differences displayed by these two domains in binding the SLP-76 RxxK motif are likely rooted in their distinctive biological functions. Gads is selectively expressed in hematopoietic cells, and in T cells it appears dedicated to coupling SLP-76 to LAT, whereas Grb2 is very widely expressed and has multiple binding partners and biological activities, including recognition of RxxK sites in other proteins, such as Gab1 and Gab2. It is also possible that some SH3 domains can bind either PxxP- or RxxK-based peptide ligands. Since the binding surfaces on the SH3 domain for these two ligand classes overlap, such interactions would be mutually exclusive. Multiple ligands with distinct binding specificities might therefore compete for the same SH3 domain, based on their local concentrations or relative affinities. In the case of Gads SH3-C domain, its high affinity for SLP-76 mediated by a novel mode of ligand recognition forms the basis for their specific interactions in T cell development and TCR signaling. It is of interest that the Gads SH3-C domain-binding site in SLP-76 is disordered prior to association with Gads. The potential for Gads to induce a structural reorganization of SLP-76 may be important in assembly of the SLP-76 multiprotein complex. Experimental Procedures Peptide Synthesis and Binding Studies Using Fluorescence Polarization Peptides were synthesized on Fmoc-amide resin (Applied Biosystems) at a 0.25 mmol scale on an AbiMed 431 synthesizer utilizing

the Fastmoc protocol. Peptide purification was accomplished by HPLC and peptide identity confirmed by MALDI mass spectrometry. Equilibrium binding constant determination and IC50 measurements were carried out using fluorescence polarization as previously described (Nash et al., 2002). Mutagenesis, Cell Culture, and Immunoprecipitation Wild-type (clone E6.1) and SLP-76-deficient (clone J14) Jurkats were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and antibiotics. SLP-76 and Gads constructs with the indicated mutations were generated by PCR and cloned into a pEFBOS vector with a Flag epitope tag. SLP-76-deficient Jurkat T cells (clone J14) were electroporated with 40 ␮g of pEF-wild-type or mutant SLP-76, and anti-Gads immunoprecipitations (IPs) were performed using 1 mg of clarified lysates as previously described (Liu et al., 1999). Immunoprecipitated SLP-76 was detected by Western blot using a monoclonal anti-Flag antibody at a 1:1000 dilution (Sigma). Gads IPs were reprobed with polyclonal anti-Gads antibody (1:500) to show equal precipitation of Gads. In a separate experiment E6.1 (SLP-76 expressing) Jurkats were electroporated with 40 ␮g of Flag-tagged Gads constructs and anti-Flag IPs were performed on 1 mg of clarified lysate. To detect SLP-76 binding, IPs were blotted with anti-SLP-76 sera (a generous gift from Gary Koretzky) and reprobed with anti-Flag antibody to show equal precipitation of the mutant Gads proteins. NMR Spectroscopy and Structure Calculation Methods for the preparation of uniformly 15N/13C-labeled Gads SH3-C domain and SLP-76 peptide were reported elsewhere (Liu and Li, 2002). Natural abundance peptide was synthesized chemically and purified to homogeneity before use (Berry et al., 2002). Two samples with different isotope labeling schemes were used. Sample #1 contained approximately 1 mM uniformly double-labeled Gads SH3-C domain mixed with unlabeled SLP-76 peptide in a 1:1 molar ratio. Sample #2 was a 1:1 complex of unlabeled Gads SH3C domain (⫺1 mM) and double-labeled SLP-76 peptide. The buffer used consisted of 50 mM sodium phosphate, 100 mM NaCl, 0.02% NaN3 (pH 6.0) in 10% D2O and 90% H2O. NMR spectra were recorded at 12⬚C on a Varian Inova 600 MHz spectrometer. Experiments used for obtaining sequence-specific resonance assignments of the protein complex have been described previously (Liu and Li, 2002). Distance restraints were derived from 3D 15N/13C-edited NOESY with a mixing time of 150 ms performed on both samples. Intermolecular distance restraints were derived from 3D half-filtered NOESY (Zwahlen et al., 1997) with a mixing time of 150 ms on sample #2. Methyl groups of valine and leucine were stereo-specifically assigned by using a similar method as reported by Neri et al. (1989). Hydrogen bond restraints in the Gads SH3-C domain were obtained from 15N-edited HSQC spectrum recorded 1 hr after deuterium exchange. Dihedral restraints were based on both the HNH␣J-coupling constants determined in a 3D HNHA experiment (Vuister and Bax, 1993) and those predicted by TALOS (Comilescu et al., 1999) using N, H␣, C␣, C␤, and C chemical shifts. Structures were calculated using the program ARIA1.0 (Niles and O’Donoghue, 1998) using the torsion angle dynamics protocol with eight iterations. A group of 20 structures were calculated in the first seven iterations, and 100 structures were calculated in the final iteration. For the final 20 structures of lowest average energy, no distance restraint violations larger than 0.25 A˚ were found. Acknowledgments This work was supported by grants from The Cancer Research Society Inc. (to S.S.-C.L.), the National Cancer Institute of Canada (NCIC), and the Canadian Institute of Health Research (CIHR) (to S.S.-C.L., C.J.M., and T.P.). S.S.-C.L. and C.J.M. are Research Scientists of NCIC with funds made available by the Canadian Cancer Society. P.N. is the recipient of a senior postdoctoral fellowship from CIHR. T.P. is a distinguished scientist of the CIHR. Received: September 18, 2002 Revised: December 2, 2002

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