High-Resolution 3D Reconstruction of a Dynamin Mutant, K44A, in its Super-Constricted State

Abstract

Dynamin assembles around the necks of budding vesicles during clathrin-mediated endocytosis and membrane trafficking in the cell. Upon GTP hydrolysis, dynamin constricts the neck, leading to membrane fission. We have previously shown that the presence of GTP drives dimerization of opposing GTPase domains in assembled dynamin polymers. This evokes a power stroke in the structure, which mediates membrane fission. Exactly how fission occurs in still unclear. Failure of dynamin to hydrolyze GTP results in block of endocytosis in the cells. To further elucidate the role of GTP hydrolysis during dynamin-mediated fission, we examined a GTP hydrolysis mutant, K44A. Here, we show that in the presence of GTP, K44A tubulates liposomes and constricts the underlying lipid to an extent far exceeding what was previously observed with other dynamin mutants. Sedimentation assays further show that, unlike wt dynamin, K44A-decoration of the lipid tube persists in the presence of GTP even after 30 minutes of incubation. In addition, we solved the 3D structure of K44A super-constricted tubes using cryo-electron microcopy and images processing methods. The 3D map revealed an inner diameter of ∼4 nm, a diameter predicted to be sufficient for spontaneous membrane fission. We have further docked the crystal structures of individual dynamin domains into our 3D map and the best fit for the GTPase domain is in the GTP state and not the GDP/AlFx transition state. This suggests that GTP binding, and not GTP hydrolysis, drives constriction of the dynamin polymer into a super-constricted pre-fission state. Following constriction, the GTP-hydrolysis-driven power-stroke may then ultimately lead to disassembly of the polymer, resulting in membrane fission.