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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

基本信息

批准号：
7734266
负责人：
Jenny E Hinshaw
金额：
$ 35.47万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/7734266
关键词：
Algorithms Alleles Arginine Cells Centronuclear myopathy Charcot-Marie-Tooth Disease Clathrin Collaborations Constriction procedure Coupled Defect Dictyostelium discoideum dynamin A Disease Docking Drosophila genus Dynamin Endocytosis Enzymes Event F-Actin Family member Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Homologous Gene Human Hydrolysis Link Lipid Bilayers Lipid Binding Lipids Liposomes Mammalian Cell Maps Mediating Membrane Membrane Protein Traffic Methods Modeling Molecular Conformation Motion Mutation Neck Nutrient PH Domain Peripheral Nervous System Diseases Play Positioning Attribute Proline Protein Family Protein Overexpression Radial Recycling Role Signal Transduction Structure Suggestion Synaptic Membranes Temperature Testing Tube Virginia Work coated pit density insight light microscopy member molecular modeling mutant platelet protein P47 radius bone structure reconstruction self assembly shibire gene product three dimensional structure tool

项目摘要

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. More recent work, using light microscopy, has confirmed our suggestion that dynamin constricts the underlying lipid bilayer. 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 first structure of dynamin in the constricted state using helical reconstruction methods and the structure of dynamin in the non-constricted state using the IHRSR method in collaboration with Dr. Edward Egelman at U. Virginia. 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 have combined structural information available from the crystal structures with the cryo-EM density to generate a model of specific dynamin interactions. We have docked the GTPase and PH domains of dynamin into the 3D maps using a rigid-body Monte Carlo algorithm. 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.

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。