Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, α-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.

SUMMARY Dynamin is a 100 kDa GTPase that organizes into helical assemblies at the base of nascent clathrin-coated vesicles. Formation of these oligomers stimulates the intrinsic GTPase activity of dynamin, which is necessary for efficient membrane fission during endocytosis. Recent evidence suggests that the transition-state of dynamin's GTP hydrolysis reaction serves as a key determinant of productive fission. Here we present the structure of a transition-state-defective dynamin mutant, K44A, trapped in a pre-fission state, at 12.5 Å resolution. This structure constricts to 3.7 nm, reaching the theoretical limit required for spontaneous membrane fission. Computational docking indicates that the ground state conformation of the dynamin polymer is sufficient to achieve this super-constricted pre-fission state and reveals how a 2-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. These data suggest a new model for the assembly and regulation of the minimal dynamin fission machine.

Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate1,2, characterized by a ‘stalk’ in which only the inner monolayers of the two compartments have merged to form a localized non-bilayer connection1-3. Formation of the hemi-fission intermediate requires energy input from proteins catalyzing membrane remodeling; however the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analyzed how the GTPase cycle of dynamin, the prototypical membrane fission catalyst4-6, is directly coupled to membrane remodeling. We used intra-molecular chemical cross-linking to stabilize dynamin in its GDP•AlF4--bound transition-state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fueled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction7-8, the force bimodality might constitute a general paradigm for leakage-free membrane remodeling.

Clathrin-mediated vesicle recycling in synapses is maintained by a unique set of endocytic proteins and interactions. We show that endophilin localizes in the vesicle pool at rest and in spirals at the necks of clathrin-coated pits (CCPs) during activity in lamprey synapses. Endophilin and dynamin colocalize at the base of the clathrin coat. Protein spirals composed of these proteins on lipid tubes in vitro have a pitch similar to the one observed at necks of CCPs in living synapses, and lipid tubules are thinner than those formed by dynamin alone. Tubulation efficiency and the amount of dynamin recruited to lipid tubes are dramatically increased in the presence of endophilin. Blocking the interactions of the endophilin SH3 domain in situ reduces dynamin accumulation at the neck and prevents the formation of elongated necks observed in the presence of GTPγS. Therefore, endophilin recruits dynamin to a restricted part of the CCP neck, forming a complex, which promotes budding of new synaptic vesicles.