title: SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity.
abstract: Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Sai
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Collett
authors.affiliation: Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, New South Wales, Australia.
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D034284:Dynamin I / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D018909:src Homology Domains
chemical_list: D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C075637:amphiphysin; D012965:Sodium Chloride; D005982:Glutathione Transferase; D034284:Dynamin I; D020558:GTP Phosphohydrolases