High Resolution Crystal Structure of the Grb2 SH2 Domain with a Phosphopeptide Derived from CD28

Kunitake Higo,Nobutoshi Ito,Jun Takahashi,Hisayuki Morii,Ryo Abe,Teikichi Ikura,Masayuki Oda,Jon C.D Houtman

doi:10.1371/journal.pone.0074482

Abstract

Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.

Highlights

Src homology 2 (SH2) domains are critical components of intracellular proteins that promote signal transduction
We report the crystal structure of Growth-factor receptor-bound protein 2 (Grb2) SH2 in complex with a CD28-derived peptide consisting of 8 amino acids, including the pY-M-N-M sequence, at a resolution of 1.35 A
The folds of the Grb2 SH2 domain were essentially the same as those previously reported; consisting of a central, antiparallel b-sheet flanked by 2 a-helices (Fig. 1A) [4,5,6,7]

Summary

Introduction

Src homology 2 (SH2) domains are critical components of intracellular proteins that promote signal transduction. SH2 domains recognize phosphotyrosine (pY)-containing sequences in proteins. Growth-factor receptor-bound protein 2 (Grb2) is an adaptor protein that has an SH3-SH2-SH3 domain architecture [1]. The Grb SH2 domain mediates activation of the Ras pathway through binding to phosphotyrosyl motifs on either growth factor receptors such as epidermal growth factor receptor or other adaptor proteins such as Shc [2]. Grb SH2 binds to the pY-X-N-X consensus sequence where X is any amino acid; it binds to pY-(L/V)-N-(V/P) with higher affinity [3,4]. The selective inhibition of Grb SH2 binding to phosphorylated proteins is expected to be useful for the prevention of hyperproliferative diseases

Methods

Results

Conclusion