title: Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.
abstract: SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.
authors.affiliation: ARIAD Pharmaceuticals, Cambridge, Massachusetts 02139, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Weng
authors.forename: R J
authors.initials: RJ
authors.lastname: Rickles
authors.forename: S
authors.initials: S
authors.lastname: Feng
authors.forename: S
authors.initials: S
authors.lastname: Richard
authors.forename: A S
authors.initials: AS
authors.lastname: Shaw
authors.forename: S L
authors.initials: SL
authors.lastname: Schreiber
authors.forename: J S
authors.initials: JS
authors.lastname: Brugge
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001224:Aspartic Acid / Q000502:physiology*; D053487:DEAD-box RNA Helicases; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D001224:Aspartic Acid; D001120:Arginine; D011392:Proline; D011494:Protein Kinases; D016315:Proto-Oncogene Proteins c-abl; D016392:Proto-Oncogene Proteins pp60(c-src); C506751:Ddx5 protein, mouse; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases