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.     

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.     

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.     

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.     

Kinetic comparison of the catalytic domains of SHP-1 and SHP-2

The phosphatase activity of SH2-containing protein tyrosine phosphatase (SHP) is inhibited by its SH2 domains and C-terminal tail. In order to determine the inhibitory effects of the SH2 domains and C-terminal tail, we have expressed and purified the catalytic domains of SHP-1 and SHP-2, and the SH2 domain truncated SHP-1 and SHP-2. We have then measured their kinetic parameters using p-nitrophenyl phosphate (p-NPP) and phosphotyrosine (pY) as substrates under the same experimental conditions. The results indicate that the pH-dependent profiles of SHP-1 and SHP-2 are mainly determined by their catalytic domains. Both enzymes have maximum activity at pH 5.0. In addition, the phosphatase activity of different forms of SHP-1 and SHP-2 decreases as the salt concentration increases. Without SH2 domains, both SHP-1 and SHP-2 are no longer inhibited by their C-terminal tails. However, the C-terminal tail of SHP-1 can further prevent the salt inhibition of the phosphatase activity. Under the same experimental conditions, the catalytic domain of SHP-1 is two times more active than the catalytic domain of SHP-2.     

Simultaneous binding of two peptidyl ligands by a SRC homology 2 domain

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing phosphotyrosine (pY)-containing sequences of target proteins. In all of the SH2 domain-pY peptide interactions described to date, the SH2 domain binds to a single pY peptide. Here, determination of the cocrystal structure of the N-terminal SH2 domain of phosphatase SHP-2 bound to a class IV peptide (VIpYFVP) revealed a noncanonical 1:2 (protein-peptide) complex. The first peptide binds in a canonical manner with its pY side chain inserted in the usual binding pocket, while the second pairs up with the first to form two antiparallel β-strands that extend the central β-sheet of the SH2 domain. This unprecedented binding mode was confirmed in the solution phase by NMR experiments and shown to be adopted by pY peptides derived from cellular proteins. Site-directed mutagenesis and surface plasmon resonance studies revealed that the binding of the first peptide is pY-dependent, but phosphorylation is not required for the second peptide. Our findings suggest a potential new function for the SH2 domain as a molecular clamp to promote dimerization of signaling proteins.     

Identification of natural ligands for SH2 domains from a phage display cDNA library

The cytoplasmic domain of the Fc gamma receptor IIB (FcgammaRIIB) can be successfully displayed on the surface of filamentous phage, and after phosphorylation in vitro, can interact specifically with the SH2 domains of SHP-2, a cytoplasmic tyrosine phosphatase. When full-length FcgammaRIIB is expressed on phage, however, this interaction is greatly compromised, illustrating that characteristics of the full-length sequence are not well tolerated by the phage display system. Many associations in cell physiology are driven by similar interactions involving small modular binding domains or ligands, and so a fragmented cDNA library will facilitate display of such domains free of sequences which compromise their expression. A fragmented leukocyte cDNA display library of 10(8) clones was constructed. This library was phosphorylated in vitro with fyn kinase and was selected against the tandem SH2 domains of SHP-2 in the search for additional ligands. A depletion strategy to remove non-specific clones was employed, using SHP-2 Sepharose, prior to in vitro phosphorylation and selection. This permitted the emergence of clones encoding the cytoplasmic domain of PECAM-1, another natural ligand for SHP-2. The importance of dual phosphorylation of tyrosine residues at positions 663 and 686 was confirmed in competition ELISA experiments using phosphorylated phage and synthetic peptides. Thus, phage display of fragmented cDNA libraries permits the identification and characterisation of phosphorylated ligands of modular binding domains based on their functional interaction.     

A model of activation of the protein tyrosine phosphatase SHP-2 by the human leptin receptor

Signalling through the leptin receptor has been shown to activate the SH2 domain-containing tyrosine phosphatase SHP-2 through tyrosine phosphorylation. The human leptin receptor contains five tyrosine residues in the cytoplasmic domain that may become phosphorylated. We show here using BIAcore studies, wherein binding of peptides to SHP-2 was detected, that peptides corresponding to sequences containing phosphotyrosines 974 and 986 (LR974P and LR986P, respectively) from the leptin receptor cytoplasmic domain were the only two peptides that bound to the enzyme. Binding of LR974P to SHP-2 was inhibited in a dose-dependent fashion by orthovanadate, whereas binding of LY986P was not, indicating that the enzyme binds to these peptides through different sites. Only the leptin receptor-derived peptide corresponding to tyrosine 974 was dephosphorylated by recombinant purified SHP-2. Time courses of the reaction were complex, and fitted a two exponent rate equation. Preincubation of SHP-2 with LR986P markedly activated the enzyme at early time points and time courses of the activated enzyme fitted a single exponential first order rate equation. We propose that LR974P binds to the active site of SHP-2, whereas LR986P may bind to the N- and C-terminal SH2 domains of SHP-2, thus activating the phosphatase activity. These data support a model in which SHP-2 binds to phosphotyrosine 986 in the activated leptin receptor and is activated to dephosphorylate phosphotyrosine 974, downregulating signalling events emanating from SH2 domain-containing proteins that bind here.     

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.     

Molecular dynamics simulations on the free and complexed N-terminal SH2 domain of SHP-2

Signal transduction events are often mediated by small protein domains such as SH2 (Src homology 2) domains that recognize phosphotyrosines (pY) and flanking sequences. In case of the SHP-2 receptor tyrosine phosphatase an N-terminal SH2 domain binds and inactivates the phosphatase (PTP) domain. The pY-peptide-binding site on the N-terminal SH2 domain does not overlap with the PTP binding region. Nevertheless, pY-peptide binding causes domain dissociation and phosphatase activation. Comparative multi-nanosecond molecular dynamics simulations on the N-SH2 domain in ligand-bound and free states have been performed to study the allosteric mechanism that leads to domain dissociation upon pY-peptide binding. Significant ligand-dependent differences in the conformational flexibility of regions that are involved in SH2-PTP domain association have been observed. The results support a mechanism of signal transduction where SH2-peptide binding modulates the domain flexibility and reduces its capacity to fit into the entrance of the PTP catalytic domain of SHP-2.     

The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 (SHP-2-(198-593)), but not the smaller SHP-2-(399-593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198-593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198-593) mutant did not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399-593) increased Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibited SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.     

Screening combinatorial libraries by mass spectrometry. 2. Identification of optimal substrates of protein tyrosine phosphatase SHP-1

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. In this work, we have developed a novel combinatorial library method, termed "enzyme-catalyzed loss of isotope peak signal enhancement (ECLIPSE)", to determine the substrate specificity of PTPs. This method involves partial labeling of pY at a nonbridging phosphate oxygen atom with 50% (18)O ((16)O/(18)O = 1:1). A 361-member solution-phase peptide library with randomization at the -1 and -2 positions (relative to pY), RNNXXpYA-NH(2) (X = 19 alpha-amino acids except for Cys), was synthesized with the partially (18)O-labeled pY by the split-synthesis method. Each member of the resulting pY peptide library appeared as a doublet peak in the mass spectrum (m/z m and m + 2.0043). Limited treatment of the library with a PTP removed the mass-degenerate phosphoryl group from the most preferred substrates to generate products as singlet peaks, which were readily identified and sequenced by tandem mass spectrometry. Screening of the pY library against the catalytic domain of SHP-1 revealed that SHP-1 prefers an acidic residue at the -2 position, with aspartic acid being slightly better than glutamic acid. At the -1 position, SHP-1 also prefers an acidic residue, although a variety of other amino acids are also tolerated. On the other hand, positively charged residues at these positions render the corresponding peptides very poor substrates of SHP-1. Several selected peptides were individually synthesized and assayed against SHP-1, and the kinetic data confirmed the screening results. These results demonstrate that ECLIPSE is a viable method for studying the substrate specificity of PTPs.     

Conformational Determinants of Phosphotyrosine Peptides Complexed with the Src SH2 Domain

The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA) to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the “two-pronged plug two-hole socket” model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an α-helix. In contrast, a β-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors.     

Binding of phosphatidic acid to the protein-tyrosine phosphatase SHP-1 as a basis for activity modulation.

Activation of the SH2 domain-possessing protein-tyrosine phosphatase SHP-1 by acidic phospholipids as phosphatidic acid (PA) has been described earlier and suggested to participate in regulation of SHP-1 activity toward cellular substrates. The mechanism of this activation is poorly understood. Direct binding of phosphatidic acid to recombinant SHP-1 could be demonstrated by measuring the extent of [(14)C]PA binding in a chromatographic assay, by measuring the extent of binding of SHP-1 to PA-coated ELISA plates or silica beads (TRANSIL), and by spectroscopic assays employing fluorescently labeled PA liposomes. In addition to PA, phosphatidylinositol 3,4, 5-trisphosphate (PIP3), dipalmitoylphosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, and phosphatidylserine (PS) were found to bind to SHP-1, albeit to a lesser extent. A high-affinity binding site for PA and PIP3 was mapped to the 41 C-terminal amino acids of SHP-1. This site was absent from the related protein-tyrosine phosphatase SHP-2 and conferred activation of SHP-1 by PA toward two different substrates at low lipid concentrations. A SHP-1 mutant missing this binding site could, however, still be activated toward phosphorylated myelin basic protein as a substrate at high PA concentrations. This activation is likely to be mediated by a second, low-affinity binding site for PA in the N-terminal part of SHP-1 within the SH2 domains. High-affinity phospholipid binding to the C-terminus of SHP-1 may present a specific mechanism of regulating activity and/or cellular localization.     

Substrate specificity of protein tyrosine phosphatases 1B, RPTPα, SHP-1, and SHP-2

We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately 2 orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active toward multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY-1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY-1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme's in vivo substrate specificity.     

SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phosphatase SHP-2 have similar binding specificities

Suppressor of cytokine signaling-3 (SOCS-3) and the protein tyrosine phosphatase SHP-2 both regulate signaling by cytokines of the interleukin-6 family, and this is dependent upon recruitment to tyrosine 757 in the shared cytokine receptor subunit gp130. To better explore the overlap in ligand binding specificities exhibited by these two signaling regulators, we have mapped the phosphopeptide binding preferences of the SH2 domains from SOCS-3 and SHP-2. Degenerate phosphopeptide libraries were screened against recombinantly produced SH2 domains to determine the sequences of optimal phosphopeptide ligands. We found that the consensus ligand binding motif for SOCS-3 was pY-(S/A/V/Y/F)-hydrophobic-(V/I/L)-hydrophobic-(H/V/I/Y), while the consensus motif for SHP-2 was pY-(S/T/A/V/I)-X-(V/I/L)-X-(W/F). We validated these data through the design of phosphopeptide ligands based on the consensus motifs and found that these bound to SOCS-3 and SHP-2 with high affinity. Finally, we have compared the affinity of SOCS-3 for binding to phosphopeptides representing putative docking sites in the gp130, leptin and erythropoietin receptors. While SOCS-3 binds with much higher affinity to a gp130 phosphopeptide than to phosphopeptides derived from the other receptors, multiple SOCS-3 binding sites are predicted to exist in the leptin and erythropoietin receptors which may compensate for weaker binding to individual sites.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.     

Solution structure of the yeast Rad53 FHA2 complexed with a phosphothreonine peptide pTXXL: comparison with the structures of FHA2-pYXL and FHA1-pTXXD complexes

It was proposed previously that the FHA2 domain of the yeast protein kinase Rad53 has dual specificity toward pY and pT peptides. The consensus sequences of pY peptides for binding to FHA2, as well as the solution structures of free FHA2 and FHA2 complex with a pY peptide derived from Rad9, have been obtained previously. We now report the use of a pT library to screen for binding of pT peptides with the FHA2 domain. The results show that FHA2 binds favorably to pT peptides with Ile at the +3 position. We then searched the Rad9 sequences with a pTXXI/L motif, and tested the binding affinity of FHA2 toward ten pT peptides derived from Rad9. One of the peptides, (599)EVEL(pT)QELP(607), displayed the best binding affinity (K(d)=12.9 microM) and the greatest chemical shift changes. The structure of the FHA2 complex with this peptide was then determined by solution NMR and the structure of the complex between FHA2 and the pY peptide (826)EDI(pY)YLD(832) was further refined. Structural comparison of these two complexes indicates that the Leu residue at the +3 position in the pT peptide and that at the +2 position in the pY peptide occupy a very similar position relative to the binding site residues from FHA2. This can explain why FHA2 is able to bind both pT and pY peptides. This position change from +3 to +2 could be the consequence of the size difference between Thr and Tyr. Further insight into the structural basis of ligand specificity of FHA domains was obtained by comparing the structures of the FHA2-pTXXL complex obtained in this work and the FHA1-pTXXD complex reported in the accompanying paper.     

Structural Determinants of SHP-2 Function and Specificity in Xenopus Mesoderm Induction

SHP-2 is a positive component of many receptor tyrosine kinase signaling pathways. The related protein-tyrosine phosphatase (PTP) SHP-1 usually acts as a negative regulator. The precise domains utilized by SHP-2 to transmit positive signals in vivo and the basis for specificity between SHP-1 and SHP-2 are not clear. In Xenopus, SHP-2 is required for mesoderm induction and completion of gastrulation. We investigated the effects of SHP-2 mutants and SHP-2/SHP-1 chimeras on basic fibroblast growth factor-induced mesoderm induction. Both SH2 domains and the PTP domain are required for normal SHP-2 function in this pathway. The N-terminal SH2 domain is absolutely required, whereas the C-terminal SH2 contributes to wild-type function. The C-terminal tyrosyl phosphorylation sites and proline-rich region are dispensable, arguing against adapter models of SHP-2 function. Although the SH2 domains contribute to SHP-2 specificity, studies of SHP chimeras reveal that substantial specificity resides in the PTP domain. Thus, PTP domains exhibit biologically relevant specificity in vivo, and noncatalytic and catalytic domains of PTPs contribute to specificity in a combinatorial fashion.