Structure And Function Of Dynamin, A 100kd GTPase Involved In Endocytosis

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. The founding member, dynamin, is crucial for endocytosis, synaptic membrane recycling, membrane trafficking within the cell and more recently, has been associated with filamentous actin. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue of the shibire gene product in Drosophila. A temperature sensitive shibire allele causes a defect in clathrin-mediated endocytosis. Since then, overexpressing human dynamin mutants in mammalian cells was found to block clathrin-mediated endocytosis. Over the years, our cryo-electron microscopy (cryoEM) structural work has played a leading role in dissecting the function of dynamin in membrane fission. We have shown that purified dynamin readily assembles into rings and spirals and it forms similar structures on liposomes, generating dynamin-lipid tubes that constrict upon GTP hydrolysis. A potential mechanism for dynamin constriction was revealed when we solved the three-dimensional structure of dynamin in the non-constricted and constricted states by cryo-electron microscopy (cryo-EM). These results suggest dynamin wraps around the necks of budding vesicles as a helical polymer and upon GTP hydrolysis undergoes a significant constriction that ultimately leads to membrane fission. In 2018, we solved a high-resolution cryo-EM structure of the membrane-associated helical polymer of human dynamin-1 in the GTP-bound state (3.75 Angstroms). Images for the high-resolution structure were collected at the New York Structural Biology Center (NYSBC) in New York City using a FEI Krios electron microscope with a K2 direct electron detector. The dynamin helical structure allowed us to build an atomic model of the assembled dynamin polymer bound to lipid. Comparing soluble crystal structures to our new high-resolution cryo-EM structure revealed conformational changes that occur upon assembly and lipid binding. The structure defines the 1-start helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays in collaboration with Dr. Justin Taraska, NHLBI. The inner lumen of the dynamin-lipid tube is 7 nm compared to 20 nm observed in the apo state. In 2020-21, we solved the structure of a GTPase-defective dynamin mutant (K44A), in the presence of GTP, to 3.6 Angstrom resolution, which allowed us to build an atomic model. The K44A structure resembles a previous low-resolution map of WT dynamin in a post-hydrolysis state (10 Angstroms). The high-resolution cryoEM density indicates dynamin is in the GDP-bound state, further constricts the underlying lipid bilayer to achieve an inner lumen of 3.4 nm and assembles as a 2-start helix. Constriction of the membrane to 3.4 nm lumen is reaching the theoretical limit required for spontaneous membrane fission, supporting the model that dynamin alone can cause membrane fission. The K44A structure also reveals how a 2-start helical symmetry promotes the most efficient packing of dynamin tetramers around the membrane neck. In addition, this year we contributed to two successful collaborations. For the first collaboration with Dr. Justin Taraska (NHLBI) we generated tomograms of unroofed cells grown on EM grids to explore the architecture of endocytic structures on the plasma membrane. As a result, we were able to show that clathrin sites maintain a constant surface area and flat lattices are loosely packed allowing for spontaneous curvature (Dev Cell, 2021). The tomograms from unroofed cells also allowed us to visualize dynamin helical structures associated with the clathrin-coated pits in vivo. We are currently comparing the in vivo dynamin helical assemblies to our K44A-dynamin high-resolution cryoEM structure. In the second collaboration with Dr. Ling-Gang Wu (NINDS), we demonstrated that dynamin assembles around large circumferences, >200nm, mimicking large necks of a novel non-coated vesicle-budding mechanism in adrenal chromaffin cells. In previous years, we collaborated with Drs. Sandra Schmid (UT Southwestern) and Vadim Frolov (U Basque Country) to explore the effect of dynamins powerstroke defined by the large swing of the BSE in dynamin. To dissect the fission reaction into stages, we utilized intra-molecular chemical cross-linking to stabilize dynamin in a conformation mimicking its transition-state. We found that dynamin trapped in the transition state is unable to mediate full fission but forms stable hemifission intermediates without phosphate release. Dynamin assembly and augmented membrane insertion of its pleckstrin homology domain drives the hemifission state. Our findings, which are consistent with molecular simulations of the fission reaction, reveal a second, unappreciated energy barrier for full fission. Thus additional conformational dynamics are required after hemifission that enable dynamin to utilize the energy of GTP hydrolysis to complete the fission reaction. Previously, we also collaborated with Drs. Sambuughin (Uniformed Services University), Goldfarb (NINDS, NIH), Renwick (Queens University, Kingston Canada), Platonov (Ammosov North-Eastern Federal University, Russian Federation) and Toro (NHGRI, NIH) to characterized a dynamin mutant that leads to a rare case of Hereditary Spastic Paraplegia (HSP). This was the first report linking a mutation in dynamin-2 to HSP. In addition, the mutation is in a region of dynamin distinct from all other dynamin-2 disease causing mutations.