A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein

Chi Hon Lee, Benjamin Leung, Mark A. Lemmon, Jie Zheng, David Cowburn, John Kuriyan, Kalle Saksela

Research output: Contribution to journalArticle

229 Scopus citations

Abstract

We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (K(D) 250 nM). In contrast to tick, affinity of the highly homologous Fyn SH3 for Nef was too weak (K(D) > 20 μM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (K(D) 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef.

Original languageEnglish (US)
Pages (from-to)5006-5015
Number of pages10
JournalEMBO Journal
Volume14
Issue number20
DOIs
StatePublished - 1995

Keywords

  • HIV
  • PxxP motif
  • SIV
  • Tyrosine kinase

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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