Alternative mode of binding to phosphotyrosyl peptides by Src homology-2 domains

Src homology-2 (SH2) domains recognize specific phosphotyrosyl (pY) proteins and promote protein-protein interactions. In their classical binding mode, the SH2 domain makes specific contacts with the pY residue and the three residues immediately C-terminal to the pY, although for a few SH2 domains, residues N-terminal to pY have recently been shown to also contribute to the overall binding affinity and specificity. In this work, the ability of an SH2 domain to bind to the N-terminal side of pY has been systematically examined. A pY peptide library containing completely randomized residues at positions -5 to -1 (relative to pY, which is position 0) was synthesized on TentaGel resin and screened against the four SH2 domains of phosphatases SHP-1 and SHP-2. Positive beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay, and the peptides were sequenced by partial Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The N-terminal SH2 domain of SHP-2 binds specifically to peptides of the consensus sequence (H/F)XVX(T/S/A)pY. Further binding studies with individually synthesized pY peptides show that pY and the five residues N-terminal to pY, but not any of the C-terminal residues, are important for binding. The other three SH2 domains also bound to the library beads, albeit more weakly, and the selected peptides did not show any clear consensus. These results demonstrate that at least some SH2 domains can bind to pY peptides in an alternative mode by recognizing only the residues N-terminal to pY.     

Defining SH2 domain and PTP specificity by screening combinatorial peptide libraries

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing short phosphotyrosyl (pY) peptide motifs in their partner proteins. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of pY proteins, counteracting the protein tyrosine kinases. Both types of proteins exhibit primary sequence specificity, which plays at least a partial role in dictating their physiological interacting partners or substrates. A combinatorial peptide library method has been developed to systematically assess the sequence specificity of SH2 domains and PTPs. A "one-bead-one-compound" pY peptide library is synthesized on 90-microm TentaGel beads and screened against an SH2 domain or PTP of interest for binding or catalysis. The beads that carry the tightest binding sequences against the SH2 domain or the most efficient substrates of the PTP are selected by an enzyme-linked assay and individually sequenced by a partial Edman degradation/mass spectrometry technique. The combinatorial method has been applied to determine the sequence specificity of 8 SH2 domains from Src and Csk kinases, adaptor protein Grb2, and phosphatases SHP-1, SHP-2, and SHIP1 and a prototypical PTP, PTP1B.     

Structural basis for specificity switching of the Src SH2 domain

The Src SH2 domain binds pYEEI-containing phosphopeptides in an extended conformation with a hydrophobic pocket, which includes ThrEF1, binding Ile(pY +3). Mutating ThrEF1 to tryptophan switches specificity to an Asn(pY +2) requirement, yielding a biological mimic of the Grb2 SH2 domain. Here we show that the Src ThrEF1Trp SH2 domain mutant binds pYVNV phosphopeptides in a beta turn conformation, which, despite differing conformations of the interacting tryptophan, closely resembles the native Grb2/pYVNV cognate peptide binding mode. The ThrEF1Trp substitution therefore switches specificity by physically occluding the pTyr +3 binding pocket and by providing additional interaction surface area for Asn(pY +2). This demonstrates structurally how novel SH2 domain specificities may rapidly evolve through single amino acid substitutions and suggests how new signaling pathways may develop.     

Molecular details of Itk activation by prolyl isomerization and phospholigand binding: the NMR structure of the Itk SH2 domain bound to a phosphopeptide

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.     

Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C‐terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β‐turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X‐ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre‐organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild‐type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide.     

Role of electrostatic interactions in SH2 domain recognition: salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase

SH2 domains are small protein domains that bind specifically to tyrosyl-phosphorylated sequences. Because phosphorylation contributes a large part of the binding free energy, it has been postulated that electrostatic interactions may play an important role in SH2 domain recognition. To test this hypothesis, we have examined the salt dependence of the interaction between tyrosyl-phosphorylated peptides and SH2 domains. The dependence of the binding constant, K(obs), on [NaCl] was shown to be strong for binding of the tandem SH2 domain of the Syk kinase (Syk-tSH2) to doubly phosphorylated peptides derived from immune-receptor tyrosine activation motifs (dpITAMs): the slopes of plots of log(K(obs)) versus log [NaCl], designated SK(obs), ranged from -2.6 +/- 0.1 to -3.1 +/- 0.2. Binding of the single SH2 domain of the Src kinase to its consensus singly phosphorylated peptide (sequence pYEEI where pY indicates a phosphotyrosine) was also highly dependent on [NaCl] with a SK(obs) value of -2.4 +/- 0.1. The ability of salt to disrupt the interactions between Syk-tSH2 and dpITAM peptides was shown to be anion-dependent with the inhibitory effect following the order: phosphate > Cl(-) > F(-). For the Syk-tSH2 system, interactions in the pY-binding pockets were shown to be responsible for a large portion of the total salt dependence: removal of either phosphate from the dpITAM peptide reduced the magnitude of SK(obs) by 40-60% and weakened binding by 2-3 orders of magnitude. Consistent with this finding, binding of the single amino acid Ac-pY-NH(2) was characterized by a large salt dependence of binding and was also dependent on the identity of the perturbing anion. The role of peptide residues C-terminal to the pY, which are implicated in determining the specificity of the phosphopeptide-SH2 domain interaction, was next probed by comparing the binding of the Src SH2 domain to a peptide containing the pYEEI sequence with that of a lower affinity variant pYAAI peptide: the magnitude of SK(obs) for the variant peptide was reduced to -1.3 +/- 0.1 as compared to -2.4 +/- 0.1 for the pYEEI peptide, indicating that in addition to pY, residues conferring peptide binding specificity contribute significantly to the salt dependence of SH2 domain binding. This study shows that electrostatic interactions play important roles not only in mediating pY recognition and binding but also in contributing to the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Diversity in peptide recognition by the SH2 domain of SH2B1

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein‐protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate‐1 and ?2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C‐terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high‐resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand‐binding interface of SH2B1 with high specificity.     

Sequence specificity of SHP-1 and SHP-2 Src homology 2 domains. Critical roles of residues beyond the pY+3 position

A combinatorial phosphotyrosyl (pY) peptide library was screened to determine the amino acid preferences at the pY+4 to pY+6 positions for the four SH2 domains of protein-tyrosine phosphatases SHP-1 and SHP-2. Individual binding sequences selected from the library were resynthesized and their binding affinities and specificities to various SH2 domains were further evaluated by SPR studies, stimulation of SHP-1 and SHP-2 phosphatase activity, and in vitro pulldown assays. These studies reveal that binding of a pY peptide to the N-SH2 domain of SHP-2 is greatly enhanced by a large hydrophobic residue (Trp, Tyr, Met, or Phe) at the pY+4 and/or pY+5 positions, whereas binding to SHP-1 N-SH2 domain is enhanced by either hydrophobic or positively charged residues (Arg, Lys, or His) at these positions. Similar residues at the pY+4 to pY+6 positions are also preferred by SHP-1 and SHP-2 C-SH2 domains, although their influence on the overall binding affinities is much smaller compared with the N-SH2 domains. A structural model was generated to qualitatively interpret the contribution of the pY+4 and pY+5 residues to the overall binding affinity. Examination of pY motifs from known SHP-1 and SHP-2-binding proteins shows that many of the pY motifs contain a hydrophobic or positively charged residue(s) at the pY+4 and pY+5 positions.     

Two phosphorylation-independent sites on the p85 SH2 domains bind A-Raf kinase.

Src homology 2 (SH2) domains mediate phosphotyrosine (pY)-dependent protein:protein interactions involved in signal transduction pathways. We have found that the SH2 domains of the 85-kDa alpha subunit (p85) of phosphatidylinositol 3-kinase (PI3 kinase) bind directly to the serine/threonine kinase A-Raf. In this report we show that the p85 SH2:A-Raf interaction is phosphorylation-independent. The affinity of the p85 C-SH2 domain for A-Raf and phosphopeptide pY751 was similar, raising the possibility that a p85:A-Raf complex may play a role in the coordinated regulation of the PI3 kinase and Raf-MAP kinase pathways. We further show that the p85 C-SH2 domain contains two distinct binding sites for A-Raf; one overlapping the phosphotyrosine-dependent binding site and the other a separate phosphorylation-independent site. This is the first evidence for a second binding site on an SH2 domain, distinct from the phosphotyrosine-binding pocket.     

Fyn phosphorylates human MAP-2c on tyrosine 67

The Src homology 3 (SH3) domain of Fyn binds to a conserved PXXP motif on microtubule-associated protein-2. Co-transfections into COS7 cells and in vitro kinase assays performed with Fyn and wild-type, or mutant MAP-2c, determined that Fyn phosphorylated MAP-2c on tyrosine 67. The phosphorylation generated a consensus sequence for the binding of the SH2 domain of Grb2 (pYSN). Pull-down assays with SH2-Grb2 from human fetal brain homogenates, and co-immunoprecipitation of Grb2 and MAP-2 confirmed the interaction in vivo, and demonstrated that MAP-2c is tyrosine-phosphorylated in human fetal brain. Filter overlay assays confirmed that the SH2 domain of Grb2 binds to human MAP-2c following incubation with active Fyn. Enzyme-linked immunosorbent assays confirmed the interaction between the SH2 domain of Grb2 and a tyrosine-phosphorylated MAP-2 peptide spanning the pY(67)SN motif. Thus, MAP-2c can directly recruit multiple signaling proteins important for central nervous system development.     

The phosphotyrosine interaction domain of Shc binds an LXNPXY motif on the epidermal growth factor receptor.

Shc is an SH2 domain protein that is tyrosine phosphorylated in cells stimulated with a variety of growth factors and cytokines. Once phosphorylated, Shc binds the Grb2-Sos complex, leading to Ras activation. Shc can interact with tyrosine-phosphorylated proteins by binding to phosphotyrosine in the context of an NPXpY motif, where pY is a phosphotyrosine. This is an unusual binding site for an SH2 domain protein whose binding specificity is usually controlled by residues carboxy terminal, not amino terminal, to the phosphotyrosine. Recently we identified a second region in Shc, named the phosphotyrosine interaction (PI) domain, and we have found it to be present in a variety of other cellular proteins. In this study we used a dephosphorylation protection assay, competition analysis with phosphotyrosine-containing synthetic peptides, and epidermal growth factor receptor (EGFR) mutants to determine the binding sites of the PI domain of Shc on the EGFR. We demonstrate that the PI domain of Shc binds the LXNPXpY motif that encompasses Y-1148 of the activated EGFR. We conclude that the PI domain imparts to Shc its ability to bind the NPXpY motif.     

A new high affinity binding site for suppressor of cytokine signaling-3 on the erythropoietin receptor.

Erythropoietin (Epo) is a hematopoietic cytokine that is crucial for the differentiation and proliferation of erythroid progenitor cells. Epo acts on its target cells by inducing homodimerization of the erythropoietin receptor (EpoR), thereby triggering intracellular signaling cascades. The EpoR encompasses eight tyrosine motifs on its cytoplasmic tail that have been shown to recruit a number of regulatory proteins. Recently, the feedback inhibitor suppressor of cytokine signaling-3 (SOCS-3), also referred to as cytokine-inducible SH2-containing protein 3 (CIS-3), has been shown to act on Epo signaling by both binding to the EpoR and the EpoR-associated Janus kinase 2 (Jak2) [Sasaki, A., Yasukawa, H., Shouda, T., Kitamura, T., Dikic, I. & Yoshimura, A. (2000) J. Biol. Chem 275, 29338-29347]. In this study tyrosine 401 was identified as a binding site for SOCS-3 on the EpoR. Here we show that human SOCS-3 binds to pY401 with a Kd of 9.5 microm while another EpoR tyrosine motif, pY429pY431, can also interact with SOCS-3 but with a ninefold higher affinity than we found for the previously reported motif pY401. In addition, SOCS-3 binds the double phosphorylated motif pY429pY431 more potently than the respective singly phosphorylated tyrosines indicating a synergistic effect of these two tyrosine residues with respect to SOCS-3 binding. Surface plasmon resonance analysis, together with peptide precipitation assays and model structures of the SH2 domain of SOCS-3 complexed with EpoR peptides, provide evidence for pY429pY431 being a new high affinity binding site for SOCS-3 on the EpoR.     

Crystal structure of the C-terminal SH2 domain of the p85alpha regulatory subunit of phosphoinositide 3-kinase: an SH2 domain mimicking its own substrate.

The binding properties of Src homology-2 (SH2) domains to phosphotyrosine (pY)-containing peptides have been studied in recent years with the elucidation of a large number of crystal and solution structures. Taken together, these structures suggest a general mode of binding of pY-containing peptides, explain the specificities of different SH2 domains, and may be used to design inhibitors of pY binding by SH2 domain-containing proteins. We now report the crystal structure to 1.8 A resolution of the C-terminal SH2 domain (C-SH2) of the P85alpha regulatory subunit of phosphoinositide 3-kinase (PI3 K). Surprisingly, the carboxylate group of Asp2 from a neighbouring molecule occupies the phosphotyrosine binding site and interacts with Arg18 (alphaA2) and Arg36 (betaB5), in a similar manner to the phosphotyrosine-protein interactions seen in structures of other SH2 domains complexed with pY peptides. It is the first example of a non-phosphate-containing, non-aromatic mimetic of phosphotyrosine binding to SH2 domains, and this could have implications for the design of substrate analogues and inhibitors. Overall, the crystal structure closely resembles the solution structure, but a number of loops which demonstrate mobility in solution are well defined by the crystal packing. C-SH2 has adopted a binding conformation reminiscent of the ligand bound N-terminal SH2 domain of PI3K, apparently induced by the substrate mimicking of a neighbouring molecule in the crystal.     

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y¹⁹⁴ impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y¹⁹⁴ on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases.Src homology 2 (SH2) domains are modular protein structures that are important for signal transduction due to their ability to bind phosphotyrosine (pY)-containing polypeptides within defined amino acid sequence motifs (1). SH2 domains are found in various signaling enzymes and adaptor proteins. Given the reversibility of protein tyrosine phosphorylation and the affinity of SH2-pY binding, the interactions of SH2 domains are inherently dynamic and diverse. Indeed, selective, transient binding to pY motifs is a key mechanism through which intracellular signaling networks are dynamically assembled, localized, and regulated. In addition to mediating protein interactions in trans, SH2 domains bind intramolecularly (2). For example, in Src family kinases (SFKs), the SH2 domain binds in cis to the phosphorylated C-terminal tail as a mechanism to constrain and thereby auto-inhibit the intervening tyrosine kinase domain (3, 4). As well, SH2 domains of cytoplasmic tyrosine kinases have been shown to affect the kinase activity of adjacent kinase domains through allosteric interactions (5). The SFKs are therefore highly regulated as a function of their SH2 domains, which exist in dynamic equilibrium between intra- and intermolecular interactions (6). Hence, as discussed by Pawson (7), the transient and diverse interactions of an SH2 domain can regulate signaling enzymes and constitutes a major mechanism of signal transduction in response to extracellular signals.The structure of the SH2 domain has been extensively characterized. At its core is a conserved antiparallel β-sheet sandwiched between two α-helices (8). SH2 domains bind phosphotyrosine-containing peptides in an extended conformation across the central β-sheet, with the pY residue inserted in a deep recognition pocket formed by conserved residues from strands βB, βC, and βD, helix αA, and the phosphate binding loop. Peptide binding specificity is determined by more variable binding surfaces on the SH2 domain, which recognize residues C-terminal to the pY residue. For the SFK SH2 domains, the three residues C-terminal to the pY residue (pY+1,+2,+3) are dominant determinants of specificity (9, 10), with the domain binding most tightly to sequences containing the motif pYEEI (11, 12). The hydrophobic pY+3 residue inserts in a deep hydrophobic specificity pocket defined by residues of the EF and BG loops (8, 13, 14). Indeed, structural analysis of the SH2 domain revealed that the configuration of the EF and BG loops is critical in dictating SH2 domain specificity by shaping the ligand-binding surface and controlling accessibility of the pY+3 binding pocket (15). Mutation of a single residue of the EF loop can drastically impact peptide binding specificity by altering the pY+3 pocket (15–17), indicating the importance of the pY+3 pocket in substrate selectivity for the SFK SH2 domains.In addition to binding pY-containing polypeptides, SH2 domains themselves may be modulated by phosphorylation. For example, phosphorylation of the Src SH2 domain at conserved Y²¹³ resulted in activation of the cognate kinase domain, possibly by impairing SH2 binding to the phosphorylated C-terminal tail (18). Similarly, phosphorylation of Lck at the equivalent SH2 residue (Y¹⁹²) generally reduced binding to pY-peptides and proteins (19). Phosphorylation at S⁶⁹⁰ in the SH2 domain of the p85α subunit of PI 3-kinase decreased its affinity for pY-containing proteins and promoted feedback inhibition of PI 3-kinase and Akt in response to cellular starvation (20). Conversely, tyrosine phosphorylation of the tensin-3 SH2 domain stimulated substrate binding and biological activity (21). Therefore, phosphorylation of SH2 domains appears to be a general mechanism for modulating their binding properties.Here, we report that Y¹⁹⁴ in the SH2 domain of the SFK Lyn, a residue conserved in SFK SH2 domains, is frequently phosphorylated in hematopoietic and other cancers. In vitro protein and peptide interactions with the Lyn SH2 domain were affected by this phosphorylation. Our results suggest that tyrosine phosphorylation of the SFK SH2 domain modulates both its binding affinity and specificity and may constitute another layer of regulation in signaling networks.     

Novel mechanism of interaction of p85 subunit of phosphatidylinositol 3-kinase and ErbB3 receptor-derived phosphotyrosyl peptides

Ligand-activated and tyrosine-phosphorylated ErbB3 receptor binds to the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase and initiates intracellular signaling. Here, we studied the interactions between the N- (N-SH2) and C- (C-SH2) terminal SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase and eight ErbB3 receptor-derived phosphotyrosyl peptides (P-peptides) by using molecular dynamics, free energy, and surface plasmon resonance (SPR) analyses. In SPR analysis, these P-peptides showed no binding to the C-SH2 domain, but P-peptides containing a phospho-YXXM or a non-phospho-YXXM motif did bind to the N-SH2 domain. The N-SH2 domain has two phosphotyrosine binding sites in its N- (N1) and C- (N2) terminal regions. Interestingly, we found that P-peptides of pY1180 and pY1241 favored to bind to the N2 site, although all other P-peptides showed favorable binding to the N1 site. Remarkably, two phosphotyrosines, pY1178 and pY1243, which are just 63 amino acids apart from the pY1241 and pY1180, respectively, showed favorable binding to the N1 site. These findings indicate a possibility that the pair of phosphotyrosines, pY1178-pY1241 or pY1243-pY1180, will fold into an appropriate configuration for binding to the N1 and N2 sites simultaneously. Our model structures of the cytoplasmic C-terminal domain of ErbB3 receptor also strongly supported the speculation. The calculated binding free energies between the N-SH2 domain and P-peptides showed excellent qualitative agreement with SPR data with a correlation coefficient of 0.91. The total electrostatic solvation energy between the N-SH2 domain and P-peptide was the dominant factor for its binding affinity.     

Structural basis for the requirement of two phosphotyrosine residues in signaling mediated by Syk tyrosine kinase

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.     

Decoding protein-protein interactions through combinatorial chemistry: sequence specificity of SHP-1, SHP-2, and SHIP SH2 domains

A general, combinatorial library method for the rapid identification of high-affinity peptide ligands of protein modular domains is reported. The validity of this method has been demonstrated by determining the sequence specificity of four Src homology 2 (SH2) domains derived from protein tyrosine phosphatase SHP-1 and SHP-2 and inositol phosphatase SHIP. A phosphotyrosyl (pY) peptide library was screened against the SH2 domains, and the beads that carry high-affinity ligands of the SH2 domains were identified and peptides were sequenced by partial Edman degradation and mass spectrometry. The results reveal that the N-terminal SH2 domain of SHP-2 is capable of recognizing four different classes of pY peptides. Binding competition studies suggest that the four classes of pY peptides all bind to the same site on the SH2 domain surface. The C-terminal SH2 domains of SHP-1 and SHP-2 and the SHIP SH2 domain each bind to pY peptides of a single consensus sequence. Database searches using the consensus sequences identified most of the known as well as many potential interacting proteins of SHP-1 and/or SHP-2. Several proteins are found to bind to the SH2 domains of SHP-1 and SHP-2 through a new, nonclassical ITIM motif, (V/I/L)XpY(M/L/F)XP, which corresponds to the class IV peptides selected from the pY library. The combinatorial library method should be generally applicable to other protein domains.     

Structure, dynamics, and binding thermodynamics of the v-Src SH2 domain: implications for drug design

SH2 domains provide fundamental recognition sites in tyrosine kinase-mediated signaling pathways which, when aberrant, give rise to disease states such as cancer, diabetes, and immune deficiency. Designing specific inhibitors that target the SH2 domain-binding site, however, have presented a major challenge. Despite well over a decade of intensive research, clinically useful SH2 domain inhibitors have yet to become available. A better understanding of the structural, dynamic, and thermodynamic contributions to ligand binding of individual SH2 domains will provide some insight as to whether inhibitor development is possible. We report the first high resolution solution structure of the apo-v-Src SH2 domain. This is accompanied by the analysis of backbone dynamics and pK(a) values within the apo- and peptide-bound states. Our results indicate that the phosphotyrosine (pY) pocket is tightly structured and hence not adaptable to exogenous ligands. On the other hand, the pocket which accommodates residues proximal and C-terminal of the pY (pY + 3) or so-called specificity determining region, is a large dynamic-binding surface. This appears to allow a high level of promiscuity in binding. Binding of a series of synthetic, phosphotyrosyl, peptidomimetic compounds designed to explore interactions in the pY + 3 pocket further demonstrates the ability of the SH2 domain to accommodate diverse ligands. The thermodynamic parameters of these interactions show dramatic enthalpy/entropy compensation. These data suggest that the v-Src SH2 domain does not have a highly specific secondary-binding site, which clearly presents a major hurdle to design selective inhibitors.