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A ‘three-pronged’ binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome

2002; Springer Nature; Volume: 21; Issue: 3 Linguagem: Inglês

10.1093/emboj/21.3.314

1460-2075

Peter M. Hwang, Chengjun Li, Massimo Morra, Jennifer Lillywhite, D.R. Muhandiram, Frank B. Gertler, Cox Terhorst, Lewis E. Kay, Tony Pawson, Julie D. Forman‐Kay, Shuncheng Li,

Complement system in diseases

Article1 February 2002free access A 'three-pronged' binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome Peter M. Hwang Peter M. Hwang Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Search for more papers by this author Chengjun Li Chengjun Li Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 Canada Search for more papers by this author Massimo Morra Massimo Morra Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Jennifer Lillywhite Jennifer Lillywhite Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 Canada Search for more papers by this author D.Ranjith Muhandiram D.Ranjith Muhandiram Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Search for more papers by this author Frank Gertler Frank Gertler Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139 USA Search for more papers by this author Cox Terhorst Cox Terhorst Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Lewis E. Kay Lewis E. Kay Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Search for more papers by this author Tony Pawson Tony Pawson Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Julie D. Forman-Kay Julie D. Forman-Kay Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Program in Structural Biology and Biochemistry, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Shun-Cheng Li Corresponding Author Shun-Cheng Li Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 Canada Search for more papers by this author Peter M. Hwang Peter M. Hwang Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Search for more papers by this author Chengjun Li Chengjun Li Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 Canada Search for more papers by this author Massimo Morra Massimo Morra Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Jennifer Lillywhite Jennifer Lillywhite Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 Canada Search for more papers by this author D.Ranjith Muhandiram D.Ranjith Muhandiram Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Search for more papers by this author Frank Gertler Frank Gertler Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139 USA Search for more papers by this author Cox Terhorst Cox Terhorst Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215 USA Search for more papers by this author Lewis E. Kay Lewis E. Kay Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Search for more papers by this author Tony Pawson Tony Pawson Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada Search for more papers by this author Julie D. Forman-Kay Julie D. Forman-Kay Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada Program in Structural Biology and Biochemistry, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8 Canada Search for more papers by this author Shun-Cheng Li Corresponding Author Shun-Cheng Li Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 Canada Search for more papers by this author Author Information Peter M. Hwang1, Chengjun Li2, Massimo Morra3, Jennifer Lillywhite2, D.Ranjith Muhandiram1,4, Frank Gertler5, Cox Terhorst3, Lewis E. Kay1,4,6, Tony Pawson4,7, Julie D. Forman-Kay1,8 and Shun-Cheng Li 2 1Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada 2Department of Biochemistry, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 Canada 3Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215 USA 4Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8 Canada 5Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139 USA 6Department of Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada 7Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada 8Program in Structural Biology and Biochemistry, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8 Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:314-323https://doi.org/10.1093/emboj/21.3.314 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The SH2 domain protein SAP/SH2D1A, encoded by the X-linked lymphoproliferative (XLP) syndrome gene, associates with the hematopoietic cell surface receptor SLAM in a phosphorylation-independent manner. By screening a repertoire of synthetic peptides, the specificity of SAP/SH2D1A has been mapped and a consensus sequence motif for binding identified, T/S-x-x-x-x-V/I, where x represents any amino acid. Remarkably, this motif contains neither a Tyr nor a pTyr residue, a hallmark of conventional SH2 domain–ligand interactions. The structures of the protein, determined by NMR, in complex with two distinct peptides provide direct evidence in support of a 'three-pronged' binding mechanism for the SAP/SH2D1A SH2 domain in contrast to the 'two-pronged' binding for conventional SH2 domains. Differences in the structures of the two complexes suggest considerable flexibility in the SH2 domain, as further confirmed and characterized by hydrogen exchange studies. The structures also explain binding defects observed in disease-causing SAP/SH2D1A mutants and suggest that phosphorylation-independent interactions mediated by SAP/SH2D1A likely play an important role in the pathogenesis of XLP. Introduction X-linked lymphoproliferative (XLP) syndrome, also known as Duncan's disease, is a rare inherited immunodeficiency condition characterized by benign or malignant proliferation of lymphocytes, histiocytosis and alterations in serum immunoglobulin concentrations (Purtilo et al., 1975, 1978). The gene altered in XLP has recently been identified (Coffey et al., 1998; Nichols et al., 1998; Sayos et al., 1998). It encodes a small protein of 128 amino acids (variously named SAP, SH2D1A and DSHP), consisting of a five amino acid N-terminal sequence, an SH2 domain and a 25 residue C-terminal tail. A number of SAP/SH2D1A mutations have been identified in XLP patients (Nichols et al., 1998; Sayos et al., 1998; Yin et al., 1999; Sumegi et al., 2000) and these fall into three broad categories: (i) micro/macro-deletions that lead to a complete or partial loss of the gene; (ii) mutations that interfere with mRNA transcription or splicing; and (iii) nonsense mutations that result in premature termination of protein synthesis or missense mutations that give rise to substitution of an amino acid conserved between the human and murine SAP/SH2D1A proteins (Morra et al., 2001a). The missense mutations are particularly interesting since, except for one case where a stop codon at the C-terminus of the protein is replaced by that for an Arg, all the other mutations occur within the boundaries of the SH2 domain of the protein and therefore directly implicate the SH2 domain in the pathogenesis of XLP. Although mRNA analysis has strongly linked the SAP/SH2D1A gene to XLP, little is known about the molecular mechanism of the disease at the protein level. Since SAP/SH2D1A lacks an effector domain (e.g. a catalytic domain), it was suggested to function as an inhibitor of other SH2 domain-mediated interactions in T and natural killer (NK) cells so as to enhance or prolong the activities of these cells (Satterthwaite et al., 1998; Sayos et al., 1998). In line with this possibility, SAP/SH2D1A was found to bind to the cytoplasmic domain of the hematopoietic surface receptor known as the signaling lymphocyte activation molecule (SLAM), a glycosylated transmembrane protein also known as CD150 (Cocks et al., 1995). This interaction, mediated by the SAP/SH2D1A SH2 domain and a tyrosine phosphorylation site (Tyr281 in SLAM), was further shown to block the recruitment of an SH2 domain-containing tyrosine phosphatase Shp2 to the same site in SLAM (Sayos et al., 1998). Intriguingly, SAP/SH2D1A associated with SLAM regardless of the phosphorylation state of Tyr281, indicating that its SH2 domain possesses a novel binding specificity, which raises questions about the role of tyrosine phosphorylation in SAP/SH2D1A function. An analogous complex was also documented between SAP/SH2D1A and 2B4, an activating NK cell receptor that shares significant sequence identity with SLAM (Tangye et al., 1999, 2000). Moreover, engagement of 2B4 by SAP/SH2D1A prevented its association with Shp2. It is, therefore, likely that SAP/SH2D1A plays similar roles in regulating the activities of T cells (via SLAM) and NK cells (via 2B4). However, contrary to the above observations, binding of SAP/SH2D1A was recently shown to be necessary to induce tyrosine phosphorylation of SLAM by facilitating the recruitment of the Src-related tyrosine kinase FynT, rather than displacing a phosphotyrosine phosphatase (PTP) that interferes with SLAM signal transduction (Latour et al., 2001). Previously, we employed a series of truncated peptides derived from sequences flanking Tyr281 in SLAM to investigate the specificity of the SAP/SH2D1A SH2 domain (Li et al., 1999). Results demonstrated that this SH2 domain is not only capable of binding to a pTyr residue that is followed by a stretch of C-terminal amino acids in a manner akin to that of a conventional SH2 domain, but it is also capable of recognizing a pTyr residue preceded by an N-terminal fragment or an unphosphorylated Tyr residue embedded within an appropriate sequence context. Based on these observations, a 'three-pronged plug that engages three binding sites' model was put forward to account for the tripartite nature of the SAP/SH2D1A–SLAM-Tyr281 peptide interaction (Li et al., 1999). This model is consistent with the crystal structures of the SAP/SH2D1A SH2 domain in a complex with two SLAM-Tyr281 peptides, one containing a pTyr residue and the other Tyr, which demonstrated that, in addition to residues C-terminal to the pTyr/Tyr, those N-terminal are also involved in binding to the SH2 domain (Poy et al., 1999). Here, we present two solution structures of this protein: one in complex with a peptide that lacks the entire sequence C-terminal to pTyr281 and the other with an unphosphorylated SLAM peptide. These structures provide compelling evidence in support of a 'three-pronged' mechanism for peptide recognition by the SAP/SH2D1A SH2 domain, which is further supported by results obtained from a SPOTs (Frank and Doring, 1988; Blankenmeyer-Menge et al., 1990) peptide binding study demonstrating that SAP/SH2D1A binds selectively to peptides with a consensus sequence motif T/S-x-x-x-x- V/I, where x denotes any amino acid. The structures, together with amide hydrogen exchange rates measured by NMR spectroscopy, also provide a rationale for the reduced binding affinities for both the phosphorylated and unphosphorylated SLAM-Y281 peptides observed for disease-causing SAP/SH2D1A missense mutants. Results The SAP/SH2D1A SH2 domain binds to peptide motifs containing no tyrosine In order to determine the specificity of the SAP/SH2D1A SH2 domain in a more comprehensive manner than previously reported (Li et al., 1999), we synthesized a set of undecamer peptides using the SPOTs method of simultaneous multiple peptide synthesis on a derivatized cellulose membrane (Frank and Doring, 1988; Blankenmeyer-Menge et al., 1990). These peptides are based on the sequence of the Tyr281 site in SLAM (KSLTIYAQVQK), which contains five amino acids each at the N- and C-terminus of the Tyr281 residue and is thus designated n-Y-c for simplicity. Specifically, each position in peptide n-Y-c was permutated to allow for a full representation of all 20 naturally occurring amino acids at that position, generating a total of 11 × 20 = 220 spots on a cellulose membrane. A series of truncated peptides with decreasing N- or C-terminal length was also synthesized, as was a group of Ala-scanning peptides derived from peptide n-pY-c, the phosphorylated version of peptide n-Y-c (Figure 1). Figure 1.Mapping the specificity of the SAP/SH2D1A SH2 domain using peptide SPOTs synthesized on cellulose membranes. The amino acid at each position of peptide n-Y-c, KSLTIYAQVQK, was permutated to all 20 naturally occurring amino acids while maintaining the sequence of the remainder of the peptide (A). N-1 and C-1 represent a series of peptides with progressive (one residue at a time) N- and C-terminal sequence truncations of peptide n-Y-c, respectively (B and C). Ala-scanning substitutions were carried out in the background of peptide n-pY-c, KSLTIpYAQVQK (D). Download figure Download PowerPoint The peptide spots on the cellulose membrane were then screened for their ability to bind to purified glutathione S-transferase (GST)–SAP/SH2D1A. Results from the screen are displayed in Figure 1, where bright (fluorescent) spots indicate positive binding of the corresponding peptides to the protein and dark spots denote weak or negative binding. To facilitate analysis of the results, each residue in peptide n-Y-c was assigned a number according to its position relative to the central Tyr, which was given a value '0' (Figure 1). As seen in Figure 1A, residues in the peptide do not play equal roles in binding to SAP/SH2D1A. While positions +4, +5, and −3 to −5, corresponding to the extreme N- and C-termini of the peptide, and position +2 could be substituted by almost any residue without adversely affecting binding, SAP/SH2D1A is highly selective for positions −2 and +3. Binding is facilitated by residues with a hydroxyl side chain, such as Thr and Ser at position −2, and residues with hydrophobic, β-branched side chains such as Val and Ile are favored at position +3. A certain degree of selectivity was also seen at positions −1 and +1. While position −1 demonstrated a proclivity for residues with large side chains, position +1 displayed a bias against bulky, hydrophobic residues. Interestingly, there is no apparent discrimination at position 0, corresponding to the central Tyr residue in peptide n-Y-c, suggesting that within the context of the full-length peptide, the Tyr residue is completely dispensable for binding. It should be pointed out that GST alone did not show detectable binding to any of the peptide spots (data not shown). Collectively, these data demonstrate that SAP/SH2D1A selectively binds to a peptide motif containing the consensus sequence T/S-x-x-x-x-V/I. The importance of residues at positions −2 and +3 is also reflected by the N- and C-terminally truncated peptides. As shown in Figure 1B, residues at the −5 to −3 positions could be deleted without compromising the affinities of the resulting peptides. However, as soon as the peptide was shortened to exclude Thr at position −2, its affinity for SAP/SH2D1A was lost. Similarly, when the C-terminal truncation reached residue Val+3, the resulting peptide was no longer active (Figure 1C). The role of a pTyr residue in binding to SAP/SH2D1A was demonstrated by the Ala-scanning peptide series (Figure 1D). In the n-pY-c peptide background, residues at either position −2 or +3 appeared to be dispensable for binding since either could be replaced by an Ala residue without significantly compromising the affinity of the resulting peptides. Together, these observations have unambiguously identified three sites or 'prongs' in the SLAM-Tyr281 peptide that mediate high-affinity binding to SAP/SH2D1A. These prongs correspond to residues at positions −2 (Thr or Ser) and +3 (Val or Ile), and to a pTyr residue at position '0'. A combination of any two of the three prongs in a peptide is both necessary and sufficient for high-affinity SAP/SH2D1A binding. Structural basis for the 'three-pronged' binding mode of the SAP/SH2D1A SH2 domain In order to understand the structural basis underlying the 'three-pronged' interaction between SAP/SH2D1A and its targets, we determined its three-dimensional (3D) solution structure in complex with two distinct peptides, n-pY (RKSLTIpYA) and n-Y-c (RKSLTIYAQVQK). Peptide n-pY was selected for structural analysis because it is representative of a novel mode of ligand recognition by an SH2 domain, unique to SAP/SH2D1A. The structure of the SAP/SH2D1A–n-pY complex offers an example of how an SH2 domain recognizes a peptide devoid of the typical contacts C-terminal to the phosphotyrosine. Peptide n-Y-c was chosen for comparative purposes, although a crystal structure of the SAP/SH2D1A SH2 domain in complex with an essentially identical peptide was recently reported (Poy et al., 1999). The C-terminal 25 residues of SAP/SH2D1A were unstructured in both complexes. The 1H–15N HSQC cross-peaks corresponding to this region of the protein were very narrow and intense, indicative of rapid and unhindered tumbling in solution (Li et al., 1999). Moreover, no intermediate- or long-range nuclear Overhauser effects (NOEs) could be observed beyond residue 105. Consequently, only residues 1–107, corresponding essentially to the SH2 domain region of the protein (residues 6–102), were included in the structure calculations. Figure 2 shows an overlay of the 20 lowest energy structures for each complex calculated using ARIA (Nilges et al., 1998), with statistics of the structures given in Table I. Figure 2.Stereoview of a superposition of the 20 lowest energy structures of SAP/SH2D1A–SLAM peptide complexes (using backbone atoms of residues 6–56). The protein (residues 6–104) is shown in blue for the n-pY complex and in red for the n-Y-c complex. Peptide n-pY is depicted in green (only residues −4 to +1 relative to the pTyr are shown) and n-Y-c in magenta (only residues −4 to +5 are shown). Download figure Download PowerPoint Table 1. Statistics for the 20 lowest energy SAP/SH2D1A–SLAM peptide complex structures SAP/SH2D1A–n-pY SAP/SH2D1A–n-Y-c R.m.s.d. from distance restraints (Å) all (3157, 3950)a 0.0083 ± 0.0001 0.0073 ± 0.001 unambiguous (1930, 2252)b 0.0080 ± 0.0002 0.0078 ± 0.0009 ambiguous (1177, 1653) 0.0082 ± 0.0003 0.0045 ± 0.0003 hydrogen bonds (50, 52) 0.0104 ± 0.001 0.0198 ± 0.001 R.m.s.d. from dihedral restraints (°) all (171, 128) 0.157 ± 0.019 0.163 ± 0.016 Deviations from idealized geometry bonds (Å) 0.0011 ± 0.00002 0.0011 ± 0.00003 angles (°) 0.272 ± 0.001 0.273 ± 0.002 impropers (°) 0.138 ± 0.003 0.154 ± 0.004 Procheck Ramachandran map analysis most favoured regions 76.3% 79.2% additional allowed regions 22.7% 19.8% generously allowed regions 1.0% 1.0% disallowed regions 0.0% 0.0% Energy (kcal/mol) van der Waals (Lennard–Jones potential) −148 ± 17 −276 ± 20 Atomic r.m.s.d. (å) from mean structurec backbone 0.20 ± 0.05 0.29 ± 0.08 heavy 0.59 ± 0.14 0.70 ± 0.17 a The number of restraints for the n-pY and n-Y-c peptide complex structures, respectively, are given in parentheses. b Of these restraints, 392 of 1930 for the n-pY complex and 507 of 2252 for the n-Y-c complex are structurally redundant. c Superposition involved protein residues 6–102 and peptide residues −4 to +1 relative to pTyr in peptide n-pY and −4 to +5 in peptide n-Y-c. As shown in Figure 3A and B, the SAP/SH2D1A SH2 domain adopts a conventional SH2 domain fold characterized by a large central β-sheet flanked by two α-helices and a small β-sheet (Cohen et al., 1995). In both complexes, the peptides assume an extended conformation. Amino acids N-terminal to Tyr in peptide n-Y-c or pTyr in peptide n-pY make almost identical contacts with the protein in the two complexes (Figures 2 and 3). The N-terminal 'prong', Thr-2, fits into a well-defined binding site formed by Arg13, Glu17, Ile51 and Thr53 (Figure 4). Thr-2 forms a meshwork of interactions with these residues through both hydrogen bonds and hydrophobic interactions. The main-chain carbonyl group and the side-chain hydroxyl of Thr-2 are within hydrogen-bonding distance with the side chains of Thr53 and Glu17, although the latter hydrogen bond may be mediated by a water molecule (Poy et al., 1999). In addition, van der Waals contacts occur between the γ-methyl group of Ile51 and Cα of Thr-2, and between side chains of Arg13 and Thr-2 (Figure 4), as demonstrated by observed NOEs. The dual polar/hydrophobic nature of interactions occurring at position −2 explains the strong preference for Thr at this position seen in the SPOTs peptide binding study. Figure 3.Structures of the SAP/SH2D1A SH2 domain in complex with peptides n-Y-c (A and C) and n-pY (B and D). The protein is shown in ribbon representations in (A) and (B) with β-strands in cyan, α-helices in red and loops in gray, and in surface diagrams in (C) and (D) with blue indicating positive and red indicating negative electrostatic potentials. The bound peptides are in green. The secondary structural elements of the protein are labeled in (A) and (B), as are residues of the peptides important for binding. This figure was generated using MolMol (Koradi et al., 1996). Download figure Download PowerPoint Figure 4.Spatial orientation of residues in the SAP/SH2D1A SH2 domain (shown in cyan) involved in binding to the Thr-2 site (in gold) of the peptides. Protein resides shown are all within 4 Å of Thr-2 in both complexes. Oxygen atoms are colored red and nitrogen blue. Dashed lines indicate hydrogen-bonding interactions. Note that, except for the hydroxyls of the two Thr residues, hydrogen atoms are omitted for clarity. Download figure Download PowerPoint The middle 'prong', the phosphotyrosine, is present in peptide n-pY, but absent in n-Y-c. Even though the binding pocket occupied by the Tyr/pTyr side chain is virtually the same in both complexes, the unphosphorylated Tyr is more mobile. While numerous NOEs can be observed between the pTyr ring protons of peptide n-pY and Arg13, Arg32, Val37, Val40, Cys42, Ile51, Thr53, Tyr54 and Arg55 of the protein, the Tyr residue in peptide n-Y-c shows a relatively limited number of NOE contacts with Arg13, Thr53 and Arg55. The increased rigidity of the pTyr ring is due mainly to hydrogen-bonding interactions between its phosphate group and the side chains of Arg32, Ser34, Ser36 and Arg55. The SPOTs peptide binding studies also demonstrate that the Tyr residue in peptide n-Y-c could be replaced by any of the remaining 19 amino acids without compromising its affinity for SAP/SH2D1A (Figure 1). These observations collectively suggest that the majority of the binding energy from the pTyr 'prong' is contributed by interactions to the phosphate group, not the aromatic ring. The C-terminal 'prong', Val+3, is present in peptide n-Y-c, but absent in n-pY. It sits in a well-defined hydrophobic pocket lined by residues from the EF (Ala66, Glu67, Thr68, Ala69 and Lys74) and BG loops (Gly93 and Ile94). (The EF loop is located between strands E and F, while the BG loop is between helix B and strand G; Figure 3A and B). In the n-Y-c peptide complex, these loops adopt an open configuration in order to accommodate the C-terminal residues of the peptide (Figure 3A). In contrast, the two loops adopt a closed configuration in the n-pY complex (Figure 3B), a structure that bears a closer resemblance to the structure of the free protein determined by X-ray crystallography (Poy et al., 1999) than to that of the SAP/SH2D1A–SLAM-pY281 peptide complex (Table II). The switch from an open to a closed state buries a significant amount of surface area, which otherwise would be exposed to the solvent, and closes off the portion of the peptide binding groove that would otherwise interact with the C-terminal 'prong' of the peptide. This is seen most vividly in Figure 3C and D. While the two loops in the open state allow for the formation of an elongated binding groove in the n-Y-c complex (Figure 3C), the EF loop in the SAP/SH2D1A–peptide n-pY complex closes over the cleft and, in effect, creates a ridge on the protein surface, which interacts with the truncated C-terminus of peptide n-pY (Figure 3D). The use of flexible regions of a protein, such as loops, to modify the ligand-binding surface reflects the highly adaptive nature of the SAP/SH2D1A SH2 domain employing an 'induced fit' mechanism. Table 2. Comparison of different SAP/SH2D1A–peptide complex structuresa determined by X-ray crystallographyb and NMRc Complex Backbone r.m.s.d. (Å) of SH2 domain Residues 6–102 Residues 6–56 n-pY (NMR) verus n-Y-c (NMR) 1.359 0.760 Free (X-ray) verus n-pY (NMR) 0.913 0.747 pY281 (X-ray) verus n-pY (NMR) 1.218 0.848 Y281 (X-ray) verus n-Y-c (NMR) 1.069 0.606 a The sequence of peptideY281 in the crystal structures was VEKKSLTIYAQVQK. In pY281, the tyrosine is phosphorylated. b See Poy et al. (1999). c Average NMR structures are used. Backbone amide accessibility probed by hydrogen exchange rates The most striking differences between the NMR structures of the two complexes are found in the C-terminal half of the protein, including the EF and BG loops. As seen in Figure 2, residues 6–56 superimpose very well. However, between residues 58 and 95, the structures of the two complexes diverge, suggesting considerable conformational flexibility. To characterize the dynamics of this region further, we conducted hydrogen exchange experiments to measure the accessibility of SAP/SH2D1A backbone amides to a solvent. Exchange of most amide hydrogens with a solvent requires a substantial opening of structure. Thus, amide exchange experiments measure larger amplitude structural fluctuations over much longer time scales than conventional NMR relaxation-based techniques. Amide exchange protection factors were calculated for both complexes and the results are shown in Figure 5A. In both complexes, residues 58–95 display lower protection factors than the rest of the SAP/SH2D1A molecule, confirming the dynamic nature of this region. The loops in this region are particularly mobile and even the secondary structure elements (strands βE and βF, and helix αB) are not well protected from the solvent. Thus, multiple conformations are possible even within a single complex. The NMR structure of the SAP/SH2D1A– peptide n-Y-c complex is virtually identical to its corresponding X-ray structure for the protein core (residues 6–56), with a backbone r.m.s.d. of 0.6 å when the two structures are superimposed (Table II). However, the r.m.s.d. increases to 1.07 å when residues 6–102 are superimposed, again suggesting considerable conformational variability between residues 58 and 95 even though both structures are in an 'open' configuration. The differences between the NMR structures and the previously determined X-ray structures (Table II) highlight the dynamic nature of this region, which have been confirmed and quantitated by hydrogen exchange measurements (Figure 5A). Amide and methyl group relaxation experiments probing fast time scale motion have also been performed, showing behavior in agreement with the hydrogen exchange results (P.J.Finerty,Jr, D.R.Muhandiram and J.D.Forman-Kay, unpublished data). Figure 5.(A) Protection factors for the SAP/SH2D1A SH2 domain in complex with peptides n-Y-c (left) and n-pY (right). Peptides are shown in green in both complexes. Residues of the protein are color coded according to their respective protection factors on a 0–10 scale (from yellow to blue with increasing protection factors). Prolines were assigned a