Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

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.

Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.

Drp1 catalyzes mitochondrial division, but the mechanisms remain elusive. The mitochondrial lipid cardiolipin stimulates Drp1 activity and supports membrane constriction. In addition, Drp1 populates two polymeric states that equilibrate via a dimeric intermediate. Dimers nucleate Drp1 reassembly on mitochondria for fission.

The ORP lipid-binding domain can contact two membranes simultaneously to facilitate sterol extraction or delivery at one membrane in response to the lipid composition of the other.

Fluid cardiolipin (CL) promotes self-assembly of Drp1, a dynamin-family GTPase involved in mitochondrial fission. Drp1 sequesters CL into condensed membrane platforms and in a GTP-dependent manner increases the propensity of the lipid to undergo a nonbilayer phase transition. CL reorganization generates local membrane constriction for fission.

Background: Drp1 oligomerization and activity is critical for mitochondrial fission. Results: GTP hydrolysis is required for Drp1 constriction of lipid bilayers. The variable domain of Drp1 regulates self-assembly and is not required for constriction of lipid bilayers. Conclusion:The core machinery of Drp1 is sufficient to mediate lipid assembly, constriction, and disassembly. Significance: Characterization of the mechanoenzymatic properties of Drp1 advances our understanding of mitochondrial fission.