The Specificity of the N-Terminal SH2 Domain of SHP-2 Is Modified by a Single Point Mutation

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Abstract
SH2 domains are small protein domains of ∼100 amino acids that bind to phosphotyrosine (pY) in the context of a specific sequence surrounding the target pY. In general, the residues C-terminal to the pY of the binding target are considered most important for defining the binding specificity, and in particular the pY + 1 and pY + 3 residues (i.e., the first and third amino acids C-terminal to the pY). However, our previous studies with the SH2 domains of the protein tyrosine phosphatase SHP-2 [Huyer, G., Li, Z. M., Adam, M., Huckle, W. R., and Ramachandran, C. (1995) Biochemistry34, 1040−1049] indicated important interactions with the pY − 2 residue as well. In the SH2 domains of SHP-2, the highly conserved αA2 Arg is replaced by Gly. A comparison of the published crystal structures of the Src SH2 domain and the N-terminal SH2 domain of SHP-2 complexed with high-affinity peptides suggested that the αA2 Gly of SHP-2 creates a gap which is filled by the side chain of the pY − 2 residue of the bound peptide. It was predicted that replacing this Gly with Arg would alter or eliminate the involvement of the pY − 2 residue in binding. The αA2 Gly → Arg mutant was constructed, and indeed, this mutant no longer required residues N-terminal to the target pY for high-affinity binding, making its specificity more like that of other SH2 domains. The αA2 Gly is clearly involved in directing the unusual requirement for the pY − 2 residue in the binding sequence of this SH2 domain, which has important implications for its in vivo targeting and specificity.