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

参与胞吞作用的 100kd GTP 酶 Dynamin 的结构和功能

• 批准号: 9553263
• 负责人: Jenny E Hinshaw
• 金额: $ 43.47万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9553263
• 关键词: Alleles; Automobile Driving; Canada; Cells; Centronuclear myopathy; Charcot-Marie-Tooth Disease; Chemicals; Clathrin; Country; Coupled; Crystallization; Data; Defect; Dictyostelium discoideum dynamin A; Dimerization; Disease; Docking; Drosophila genus; Dynamin; Dynamin 2; Electrons; Endocytosis; Enzymes; Event; F-Actin; Family member; GTP Binding; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Spastic Paraplegia; Homologous Gene; Human; Hydrolysis; Link; Lipid Bilayers; Lipid Binding; Lipids; Liposomes; Location; Mammalian Cell; Maps; Mediating; Membrane; Methods; Microscope; Modeling; Molecular; Molecular Conformation; Movement; Mutation; National Human Genome Research Institute; National Institute of Neurological Disorders and Stroke; Neck; New York; New York City; Nutrient; PH Domain; Peripheral Nervous System Diseases; Play; Polymers; Power stroke; Protein Family; Radial; Reaction; Recycling; Reporting; Resolution; Role; Services; Signal Transduction; Structure; Synaptic Membranes; Synaptic Vesicles; Temperature; Testing; Tube; United States National Institutes of Health; Universities; Work; base; coated pit; constriction; crosslink; density; detector; dimer; disease-causing mutation; falls; improved; inorganic phosphate; insight; member; mutant; overexpression; predictive modeling; reconstruction; shibire gene product; simulation; structural biology; three dimensional structure; trafficking

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. The current model predicts dynamin wraps around the necks of coated pits and upon GTP hydrolysis constricts the necks and falls off leading to membrane fission. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. A potential mechanism for dynamin constriction was revealed when we solved the first three-dimensional structure of dynamin. We previously solved the structure of a dynamin mutant (lacking its C-terminus) in the constricted and non-constricted states 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 interior shows a large conformational change within the middle layer, which provides a clue to the mechanism of constriction. Previously, we solved the structure of Delta-PRD-dynamin and docked GMP-PCP GG domain (GTPase domain-GED fragment) crystal structure into our 3D map as well as the stalk domain from another dynamin family member, MxA, and the PH domain from dynamin. Based on the docking we predicted the location of the dimer-dimer interface. Comparison between the GG domains in the GTP-bound and transition states, suggests that the conformational change induced by the GTP hydrolysis is driving a large swing of a 3-helical bundle near the GTPase core. We predict that the helical bundle movement is dynamins power stroke that results in a significant twist and constriction of the underlying lipid bilayer leading to membrane fission. More recently we solved the structure of a transition-state-defective dynamin mutant that 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. This past year we greatly improved the resolution of the dynamin polymer by collecting data at the New York Structural Biology Center in New York City using a FEI Krios microscope with a K2 direct electron detector. Our new dynamin helical map has a resolution of approximately 4 Angstrom, which allowed us to build a 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. 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 located in a region of dynamin distinct from all other dynamin 2 disease causing mutations.

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。