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

Dynamin蛋白家族由独特的GTP酶组成，参与整个细胞的膜分裂和融合事件。创始成员Dynamin对内吞作用、突触膜循环和细胞内的膜运输至关重要，最近还与丝状肌动蛋白有关。Dynamin最初与内吞作用有关，当时它被发现是果蝇shibire基因产物在哺乳动物中的同源物。温度敏感的Shibire等位基因导致了网状蛋白介导的内吞作用的缺陷。从那时起，在哺乳动物细胞中过表达人动力素突变体被发现可以阻断笼蛋白介导的内吞作用。 多年来，我们的结构工作在剖析动力素在膜分裂中的功能方面发挥了主导作用。我们已经证明，纯化的动力素很容易组装成环和螺旋，它在脂质体上形成类似的结构，产生在GTP水解时收缩的动力素-脂管。最近的工作，使用光学显微镜，已经证实了我们的建议，即动力素收缩潜在的脂质双层。当我们解决了动力素的第一个三维结构时，我们揭示了动力素收缩的潜在机制。所有证据都支持这样一种假设，即动力素聚集在覆盖着笼蛋白的凹坑的颈部，在那里它有助于膜的分裂。动力蛋白收缩被涂层凹坑的颈部所产生的张力可能足以使细胞内的膜分裂。作为一种机械力化学酶，动力蛋白能够收缩并产生作用于底层脂双层的力量，使其在GTP酶中独一无二。 在此之前，我们与弗吉尼亚大学的Edward Egelman博士合作，使用螺旋重建方法解决了紧缩态Dynamin的第一个结构，并使用IHRSR方法解决了非紧缩态Dynamin的结构。3D体积显示了三个不同的径向密度，外层、中层和内层。在收缩过程中，最明显的变化是轴向重复次数和半径的减少。然而，体积内部在中间层显示出较大的构象变化，这为收缩机制提供了线索。Dynamin包含5个可识别的结构域：GTPase、MID、Pleckstrin同源(PH)、GTPase效应域(GED)和富含Pro/Arg的结构域(PRD)。使用分子建模工具，我们将晶体结构中可用的结构信息与低温电磁密度相结合，以生成特定动力素相互作用的模型。我们使用刚体蒙特卡罗算法将Dynamin的GTPase和PH结构域对接到3D映射中。然后，GED将驻留在中间层，这与先前的发现相吻合，即GED直接以反式与GTPase结构域相互作用，以刺激Dynamin的GTPase活性。结果表明，相邻的GTP酶结构如何以与冷冻-EM结构一致的排列相互结合，并提出了一种在收缩过程中自组装和开塞螺旋运动的机制。PH结构域在内径向密度内的定位使可变环面向膜。这一定位与PH域中对脂质结合有影响的Charcot-Marie-Tooth突变是一致的。

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.