Sequence Specificity of SHP-1 and SHP-2 Src Homology 2 Domains: CRITICAL ROLES OF RESIDUES BEYOND THE pY+3 POSITION

Diana Imhof,Anne-Sophie Wavreille,Andreas May,Martin Zacharias,Susheela Tridandapani,Dehua Pei

doi:10.1074/jbc.m601047200

Diana Imhof, Anne-Sophie Wavreille + Show 4 more

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https://doi.org/10.1074/jbc.m601047200

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Abstract

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.

Highlights

A combinatorial phosphotyrosyl peptide library was screened to determine the amino acid preferences at the pY؉4 to pY؉6 positions for the four Src homology 2 (SH2) domains of protein-tyrosine phosphatases SHP-1 and SHP-2
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
(pY to pYϩ3) are the only determinants of SH2 domain-pY peptide interaction [31, 32]. Previous work from this and other laboratories has demonstrated that residues N-terminal to pY and C-terminal to pYϩ3 can have a dramatic effect on domain binding, two modeling strategies were applied, both the binding affinity of pY peptides to several SH2 domains (10, starting from the crystal structure of SHP-2 N-SH2 domain in 14 –21, 33–35)

Summary

Introduction

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. Unlike the classical SH2 domains, SHP-1 and SHP-2 SH2 domains require amino acid residues both N- and C-terminal to pY (positions pYϪ2 to pYϩ3) for high affinity binding. Previous studies with SHP-1 showed a similar involvement of the C-terminal residues outside the region pYϪ2 to pYϩ3 for high affinity binding [10, 21]. The results show that the two N-SH2 domains strongly prefer a large hydrophobic or positively charged residue at these positions, whereas the C-SH2 domains have broader specificity

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