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

动力蛋白家族由独特的gtpase组成，参与整个细胞的膜裂变和融合事件。动力蛋白是细胞内吞作用、突触膜循环、细胞内膜运输的重要组成部分，最近又与丝状肌动蛋白联系在一起。Dynamin在果蝇中被发现是shibire基因产物的哺乳动物同源物时，首次涉及到内吞作用。温度敏感的shibire等位基因导致网格蛋白介导的内吞作用缺陷。从那时起，在哺乳动物细胞中过度表达人动力蛋白突变体被发现可以阻断网格蛋白介导的内吞作用。

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 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 first three-dimensional structure of dynamin. All evidence supports the hypothesis that dynamin assembles around the necks of clathrin-coated pits where it assists in membrane fission. The tension created by dynamin constricting the neck of coated pits may be sufficient for membrane fission in the cell. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. Previously, we solved the structure of a dynamin mutant (lacking its C-terminus) in the constricted and non-constricted states using using helical reconstruction and the IHRSR methods. The 3D volumes reveal three distinct radial densities, outer, middle and inner layers. During constriction the most obvious change is a decrease in the axial repeat and radius. However, the volume interiors shows a large conformational change within the middle layer, which provides a clue to the mechanism of constriction. Dynamin contains five identifiable domains: GTPase, middle, pleckstrin homology (PH), GTPase effector (GED) and proline/arginine-rich (PRD). Using molecular modeling tools, we docked the crystal structures of the GTPase and PH domains of dynamin into the 3D maps using a rigid-body Monte Carlo algorithm. The GTPase domain docked into the outer radial density while the PH domain docks into the inner radial density. The GED would then reside in the middle layer, which fits with previous findings that GED directly interacts in trans with a GTPase domain to stimulate the GTPase activity of dynamin. The results show how adjacent GTPase structures associate with one another in an arrangement consistent with the cryo-EM structure and suggest a mechanism for self-assembly and corkscrew motion during constriction. The positioning of the PH domain within the inner radial density places the variable loops facing toward the membrane. This positioning is consistent with the Charcot-Marie-Tooth mutation in the PH domain having an effect on lipid binding. Recently, we have solved the structure of full-length dynamin in the constricted and non-constricted states. Surprisingly, the C-terminal PRD significantly changed the packing of the dynamin dimer in the dynamin-lipid tube. In addition, we have used immunogold labeling to confirm the location of dynamin domains within the dynamin tubes. In agreement with the molecular modeling, antibodies against the GTPase domain decorate the outer surface of the dynamin tubes. Antibodies against the C-teminus also decorated the outer surface suggesting the PRD is surface exposed and accessible for binding to several of dynamin partners.