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: 8349873
• 负责人: Jenny E Hinshaw
• 金额: $ 62.75万
• 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/8349873
• 关键词: Algorithms; Alleles; Arginine; Cells; Centronuclear myopathy; Charcot-Marie-Tooth Disease; Clathrin; Complex; 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; Microinjections; Molecular Conformation; Molecular Models; Motion; Mutation; Neck; Nutrient; PH Domain; Peptides; Peripheral Nervous System Diseases; Play; Positioning Attribute; Proline; Protein Family; Proteins; Radial; Recruitment Activity; Recycling; Retrieval; Role; SH3 Domains; Signal Transduction; Structure; Synapses; Synaptic Membranes; Synaptic Vesicles; Temperature; Testing; Tube; Vesicle; Work; coated pit; constriction; density; insight; member; molecular modeling; mutant; overexpression; platelet protein P47; reconstruction; self assembly; shibire gene product; synaptojanin; three dimensional structure; tool

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 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. During synaptic membrane retrieval, dynamin and endophilin are recruited to the necks of clathrin-coated pits and play a crucial role in vesiculation. Recently we have shown that endophilin and dynamin co-localize at the necks of clathrin-coated pits, proximal to the coat, after microinjection of GTPγS. The interaction between dynamin and endophilin was further shown to be important for endocytosis by microinjection studies using the SH3-domain of endophilin and the synaptojanin derived peptide (PP19). Both perturbed the assembly of the proteins and inhibited endocytosis. In support of the role of endophilin enhancing the assembly of dynamin, we found that addition of endophilin increased the recruitment of dynamin to liposomes and the formation of tubules. These results suggest that endophilin and dynamin form a "pre-fission complex" at the necks of the coated pits proximal to the clathrin-coat, which coordinates dynamin-mediated budding of newly formed vesicles in vertebrate synapses.

发动蛋白家族的蛋白质由参与整个细胞的膜分裂和融合事件的独特GTP酶组成。 发动蛋白是细胞内吞作用、突触膜再循环、细胞膜运输的关键，最近与丝状肌动蛋白有关。 当发现发动蛋白是果蝇中shibire基因产物的哺乳动物同源物时，发动蛋白首先与内吞作用有关。 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表达人发动蛋白突变体被发现阻断网格蛋白介导的内吞作用。 多年来，我们的结构工作在解剖发动蛋白在膜分裂中的功能方面发挥了主导作用。 我们已经表明，纯化的动力蛋白容易组装成环和螺旋，它形成类似的结构上的脂质体，产生动力蛋白脂质管，收缩后GTP水解。当我们解决了发动蛋白的第一个三维结构时，揭示了发动蛋白收缩的潜在机制。所有的证据都支持这一假设，即发动蛋白聚集在网格蛋白包被的凹坑的颈部，在那里它有助于膜分裂。由发动蛋白收缩被膜纹孔的颈部所产生的张力可能足以使细胞中的膜分裂。发动蛋白收缩并对下面的脂质双层产生力的能力使其在作为机械化学酶的GTP酶中是独特的。 以前，我们解决了结构的发动蛋白突变体（缺乏其C-末端）在收缩和非收缩状态下使用螺旋重建和IHRSR方法。 三维体积揭示了三个不同的径向密度，外层，中间层和内层。 在收缩过程中，最明显的变化是轴向重复和半径的减少。 然而，体积内部显示出中间层内的大的构象变化，这为收缩机制提供了线索。 发动蛋白包含五个可识别的结构域：GT3、中间、普列克底物蛋白同源性（PH）、GT3效应子（GED）和富含脯氨酸/脯氨酸（PRD）。使用分子建模工具，我们对接到3D地图使用刚体蒙特卡罗算法的GTdR和PH域的发动蛋白的晶体结构。 GT3结构域对接到外部径向密度中，而PH结构域对接到内部径向密度中。 然后GED将驻留在中间层，这符合先前的发现，即GED直接与GTdR结构域反式相互作用以刺激发动蛋白的GTdR活性。 结果表明，如何相邻的GTbands结构与彼此的安排一致的冷冻EM结构，并建议在收缩过程中的自组装和螺旋运动的机制。 PH结构域在内部径向密度内的定位使可变环面向膜。这种定位与PH结构域中的Charcot-Marie-Tooth突变对脂质结合具有影响一致。 在突触膜修复过程中，发动蛋白和内啡肽被募集到网格蛋白包被的小凹的颈部，并在囊泡形成中发挥关键作用。 最近，我们已经表明，亲内啡肽和发动蛋白共定位在颈部网格蛋白包被的小凹，近端的外套，后微量注射GTP S。 发动蛋白和endophilin之间的相互作用被进一步证明是重要的内吞作用的显微注射研究使用的SH 3-结构域的endophilin和synaptojanin衍生肽（PP 19）。 两者都扰乱了蛋白质的组装并抑制了内吞作用。在支持的作用，内啡肽增强组装的发动蛋白，我们发现，除了内啡肽增加募集的发动蛋白脂质体和小管的形成。 这些结果表明，内嗜蛋白和发动蛋白形成一个“分裂前复合物”在颈部的涂层坑近网格蛋白外套，协调发动蛋白介导的出芽新形成的囊泡在脊椎动物突触。