The phosphopeptide-binding specificity of Src family SH2 domains

Gillian Payne, Lesley A. Stolz, Dehua Pei, Hamid Band, Steven E. Shoelson, Christopher T. Walsh

Research output: Contribution to journalArticlepeer-review

33 Scopus citations


Background: Src homology 2 (SH2) domains mediate protein/protein interactions by binding phosphotyrosyl proteins with high specificity. The protein Lck, a Src-like lymphocyte-specific tyrosine kinase which is important in signals involved in T -cell development, contains one such domain. The crystal structure of a complex of the Lck SH2 domain with a high-affinity ligand, pY324, is known. This ligand has the sequence EPQpYEEIPIYL. Results: We designed and synthesized a series of phosphopeptides with single amino-acid changes in the four residues C-terminal to the phosphotyrosine (pTyr) in pY324. Surprisingly, the Glu one residue C-terminal to the phosphotyrosine (at position pY+1) is sensitive to substitution, whereas the Ile at position pY+3 is much less sensitive, accommodating a Glu with only modest loss of binding affinity. Replacement of the Glu and Pro on either side of the Ile had little effect, as predicted. Truncated phosphopeptides that end at position pY+5 and have only an acetyl group N-terminal to the pTyr bound with only slightly lower affinity than pY324. In addition, naturally occurring phosphopeptide sequences that span a 1 000-fold range in binding affinity for the Lck SH2 domain have been identified. Conclusions: The Lck SH2 domain is highly selective for phosphotyro syl-peptide binding; its specificity is dictated by the first and third residues C-terminal to the pTyr.The unexpected effects of some amino-acid substitutions indicate that the interactions seen between SH2 domains and ligand in the crystal structure may not be identical to those that occur in solution.

Original languageEnglish (US)
Pages (from-to)99-105
Number of pages7
JournalChemistry and Biology
Issue number2
StatePublished - Oct 1994
Externally publishedYes


  • Lck
  • SH2
  • affinity
  • phosphopeptides
  • surface plasmon resonance

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry


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