title: Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation.
abstract: The amphiphysins are brain-enriched proteins, implicated in clathrin-mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, we have solved the crystal structure of the amphiphysin-2 (Amph2) SH3 domain to 2.2 A. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline-rich binding motif to which it binds on dynamin. We demonstrate that the interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self-assembly into rings. Deletion of a unique insert in the n-Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n-Src loop of the N-terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n-Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. Our data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis.
authors.affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Owen
authors.forename: P
authors.initials: P
authors.lastname: Wigge
authors.forename: Y
authors.initials: Y
authors.lastname: Vallis
authors.forename: J D
authors.initials: JD
authors.lastname: Moore
authors.forename: P R
authors.initials: PR
authors.lastname: Evans
authors.forename: H T
authors.initials: HT
authors.lastname: McMahon
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D034281:Dynamins; D004705:Endocytosis; D005347:Fibroblasts; D051380:GRB2 Adaptor Protein; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D051381:Rats; D014168:Transferrin / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D009419:Nerve Tissue Proteins; D011506:Proteins; D014168:Transferrin; C075637:amphiphysin; D020558:GTP Phosphohydrolases; D034281:Dynamins