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

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。 最近，我们已经解决了在收缩和非收缩状态下的全长动力蛋白的结构。 令人惊讶的是，C-末端PRD显著改变了动力蛋白-脂质管中动力蛋白二聚体的包装。 此外，我们已经使用免疫金标记来确认发动蛋白管内发动蛋白结构域的位置。 与分子建模一致，针对GTdR结构域的抗体装饰发动蛋白管的外表面。 针对C-末端的抗体也装饰外表面，表明PRD是表面暴露的，并且可用于与几种发动蛋白伴侣结合。

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.