Mitochondria are dynamic organelles that undergo cycles of fission and fusion. The yeast dynamin-related protein, Dnm1, has been localized to sites of mitochondrial division. Using cryo-electron microscopy (cryo-EM), we have determined the three-dimensional structure of Dnm1 in a GTP-bound state. The 3D map reveals a unique helical assembly for Dnm1 when compared with dynamin, a protein involved in vesicle scission during endocytosis. We also show that upon GTP hydrolysis Dnm1 constricts liposomes and subsequently dissociates from the lipid bilayer. The magnitude of Dnm1 constriction is substantially larger than the decrease in diameter previously reported for dynamin. We postulate that the larger conformational change is mediated by a flexible Dnm1 structure that has limited interaction with the underlying bilayer. Together, our structural studies support a mechanochemical role for Dnm1 during mitochondrial division.

Summary The GTPase dynamin catalyzes membrane fission. Though this process requires dynamin assembly, G domain dimerization and stimulated GTP hydrolysis, the underlying structural interactions and conformational changes remain a mystery. Here we present the GMPPCP-bound structures of the truncated human dynamin 1 helical polymer at 12.2Å and a fusion protein linking human dynamin 1’s catalytic G domain to its GTPase effector domain (GG) at 2.2Å. Newly resolved density features in the polymer reconstruction and the unique conformation of GGGMPPCP allowed us to position crystallized dynamin fragments in the assembled structure and define their connectivity. The resulting model shows that G domain dimers only form between tetramers in sequential rungs of the dynamin helix. Using chemical crosslinking, we demonstrate that dynamin tetramers are dimers of domain-swapped dimers. Structural comparison of GGGMPPCP to the GG transition-state complex identifies a hydrolysis-dependent powerstroke that may play a role in membrane remodeling events necessary for fission.

Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles. Here we report a 3.75 Å resolution cryo-electron microscopy structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays. Compared to the lipid-free tetramer form, membrane-associated dynamin binds to the lipid bilayer with its pleckstrin homology domain (PHD) and self-assembles across the helical rungs via its guanine nucleotide-binding (GTPase) domain. Notably, interaction with the membrane and helical assembly are accommodated by a severely bent bundle signalling element (BSE), which connects the GTPase domain to the rest of the protein. The BSE conformation is asymmetric across the inter-rung GTPase interface, and is unique compared to all known nucleotide-bound states of dynamin. The structure suggests that the BSE bends as a result of forces generated from the GTPase dimer interaction that are transferred across the stalk to the PHD and lipid membrane. Mutations that disrupted the BSE kink impaired endocytosis. We also report a 10.1 Å resolution cryo-electron microscopy map of a super-constricted dynamin polymer showing localized conformational changes at the BSE and GTPase domains, induced by GTP hydrolysis, that drive membrane constriction. Together, our results provide a structural basis for the mechanism of action of dynamin on the lipid membrane.

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.

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The dynamin family of proteins are required for numerous membrane fission and remodeling events throughout the eukaryotic cell. Dynamin is involved in the final stages of fission during clathrin‐mediated endocytosis, caveolae internalization, and vesiculation from the Golgi and recycling endosome, while Drp1 (dynamin‐related protein 1) is necessary for mitochondrial division. During membrane fission, dynamin or Drp1 is believed to wrap around the constricted site to facilitate vesiculation or organelle division. In support of this model purified dynamin has been shown to self‐assemble into spirals (50 nm diameter) and readily form dynamin‐lipid tubes, which constrict and fragment upon addition of GTP. The three‐dimensional structures of dynamin in the constricted and non‐constricted states were solved by cryo‐electron microscopy methods. Placement of the GTPase and Pleckstrin Homology crystal structures into the cryo‐EM densities revealed a twisting motion that suggests a corkscrew model for dynamin constriction. We are currently examining the structure and function of the Drp1 homologue in yeast, Dnm1, to determine if a common mechanism of action exists among dynamin family members. In collaboration with Dr. Jodi Nunnari (UC Davis), we have shown that Dnm1 assembles into large spirals with a diameter of ~110 nm, which is a similar diameter to the observed mitochondrial constriction sites seen in vivo. Dnm1 also assembles onto lipid and forms Dnm1 decorated tubes that constrict significantly upon GTP addition. A preliminary 3D map of Dnm1‐lipid tubes reveals a slightly different architecture to dynamin. For example, the Dnm1 map lacks a strong inner radial density near the lipid bilayer, which correlates well with the absence of a pleckstrin homology domain in Dnm1. Overall, these results suggest that although dynamin family members share common mechanochemical properties, the structure of each member vary to fit their unique function.