Shunming Fang

Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission

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-EM, we have determined the three-dimensional (3D) structure of Dnm1 in a GTP-bound state. The 3D map showed that Dnm1 adopted a unique helical assembly when compared with dynamin, which is involved in vesicle scission during endocytosis. Upon GTP hydrolysis, Dnm1 constricted liposomes and subsequently dissociated from the lipid bilayer. The magnitude of Dnm1 constriction was 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. Our structural studies support the idea that Dnm1 has a mechanochemical role during mitochondrial division.

A Pseudoatomic Model of the Dynamin Polymer Identifies a Hydrolysis-Dependent Powerstroke

The GTPase dynamin catalyzes membrane fission by forming a collar around the necks of clathrin-coated pits, but the specific structural interactions and conformational changes that drive this process remain a mystery. We present the GMPPCP-bound structures of the truncated human dynamin 1 helical polymer at 12.2 Å and a fusion protein, GG, linking human dynamin 1s catalytic G domain to its GTPase effector domain (GED) at 2.2 Å. The structures reveal the position and connectivity of dynamin fragments in the assembled structure, showing that G domain dimers only form between tetramers in sequential rungs of the dynamin helix. Using chemical crosslinking, we demonstrate that dynamin tetramers are made of two dimers, in which the G domain of one molecule interacts in trans with the GED of another. Structural comparison of GG(GMPPCP) to the GG transition-state complex identifies a hydrolysis-dependent powerstroke that may play a role in membrane-remodeling events necessary for fission.

Cryo-EM of the dynamin polymer assembled on lipid membrane

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 vesicles1. 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 form2, 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) domain3. 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.A cryo-electron microscopy structure of human dynamin-1 demonstrates conformational changes and sheds light on the fission of membranes during endocytosis.

A Dynamin Mutant Defines a Super-Constricted Pre-Fission Step

Dynamin is a 100 kDa GTPase that assembles around the necks of invaginated clathrin-coated pits to catalyze membrane fission during the final stages of clathrin-mediated endocytosis. Purified dynamin assembles into helical arrays on lipid templates that resemble the dense collars observed at the necks of clathrin-coated pits in vivo. Recent evidence suggests that the GTP transition state of dynamin serves as a key determinant of productive fission. The transition state stabilizes G domain dimerization, which optimally positions the catalytic machinery and thereby enhancing its intrinsic GTP hydrolysis rate. In the helical array, G domain dimerization between dynamin molecules only occurs between neighboring rungs of the helix. Thus, the architecture of the dynamin polymer ensures that assembly and stimulated turnover are tightly coupled. Here we present a three dimensional structure of a transition-state-defective mutant in the penultimate fission status at 13.5 A resolution. This structure is tightly constricted with an inner luminal diameter of 4 nm, reaching the theoretical limit required for spontaneous fission. Computational docking of dynamin crystal structures into the 3D reconstruction suggests that a GTP ground state, and not stimulated GTP hydrolysis, drives the dynamin polymer into the super-constricted pre-fission state. Computational docking also positions the proline-rich domain (PRD) close to the G domain, which supports the notion that the PRD can modify the GTPase cycle. The surface accessibility of the PRD allows dynamin partners to bind dynamin throughout its GTPase cycle, and regulate assemble, fission and disassembly.

Molecular Architecture of OPA1, the Dynamin-Related GTPase Involved in Mitochondrial Fusion

Mitochondria are dynamic organelles that constantly undergo fusion and fission events. Four dynamin-related GTPases have been shown to facilitate mitochondrial dynamics. One of them, OPA1, mediates mitochondrial inner membrane fusion. Mutations in OPA1 have been linked to severe disease phenotypes characterized by progressive degeneration of the retinal ganglion cells, thus leading to blindness. OPA1 is a nuclear encoded protein that is inserted into the inner mitochondrial membrane through an N terminal transmembrane domain. There it undergoes proteolytic processing yielding a long, membrane-bound (OPA1) and a short, diffusible form (OPA1s). Fusion of the inner mitochondrial membrane requires both forms of OPA1, and the ability of OPA1 to bind and hydrolyze GTP. In previous structural studies, we have shown that recombinant OPA1s binds to lipid preparations with a composition similar to that of the inner mitochondrial membrane (Ban et al., 2010). Our findings indicated that the interaction of OPA1s with suitable membranes stimulates oligomerization and self-assembly. Using cryo-electron microscopy, we have visualized and analyzed the topological arrangement of OPA1 on these tubules and showed that the protein wraps around the perimeter of these tubes in a helical fashion. Interestingly, the protein formed a two-dimensional lattice on the lipid bilayer indicating that specific intermolecular interactions are at play. We are now investigating the molecular architecture of such OPA1-lipid assemblies using biochemical and high-resolution electron microscopy (EM) methods. Here, we report on strategies employed to generate substrates suitable for EM analysis and on our current insights into the properties and architecture of OPA1 assemblies.Reference:Ban, T., Heymann, J. A., Song, Z., Hinshaw, J. E., Chan, D. C.: OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation. Hum. Mol. Genet. 2010, 19: 2113-22.

An Improved Model for Dynamin Assembly Revealed by Cryo-EM

Dynamin is a multidomain GTPase that assembles into collar-like structures at the necks of deeply invaginated coated pits during the final stages of clathrin-mediated endocytosis (CME) and catalyzes membrane scission. Assembly of purified dynamin tetramers in vitro yields helical structures comparable to those observed in vivo. The formation of these oligomers stimulates dynamins basal GTP hydrolysis >100-fold. Mutational analysis indicates that dynamins stimulated GTP hydrolysis is required for CME; however, mounting evidence suggests that this activity causes disassembly of the dynamin collar rather than direct membrane severing. Despite recent structural studies showing that stimulated hydrolysis arises from the transition-dependent dimerization of dynamins catalytic G domains, little is known about the conformational changes that precede and/or result from this interaction in the context of the polymer. Specifically, it is unclear how the G domains are properly oriented, which subunits associate, and how catalysis triggers dissociation of the pleckstrin homology (PH) domain at the membrane surface. Much of this ambiguity can be attributed to the low resolution (>20A) of previous dynamin polymer models and the absence of a complete dynamin tetramer crystal structure. To clarify these issues, we have used cryo-EM and iterative helical real space refinement to generate an 11A reconstruction of a truncated form of dynamin (ΔPRD) in the assembled, GMPPCP-bound state. This map reveals new structural characteristics including a twisted, interlacing interaction that stabilizes the middle/GED stalk and a previously uncharacterized density feature adjacent to the exterior GTPase head. Computational docking of crystallized dynamin fragments reveals the location and connectivity of different domains within the assembled polymer. Chemical crosslinking experiments also provide new insights into the architecture and organization of dynamin tetramer. These data have important implications regarding the conformational changes associated with dynamin catalyzed GTP hydrolysis and membrane fission.