Structure And Function Of Dynamin, A 100kd GTPase Involv

真核细胞内膜转运的动态过程涉及大量的特殊蛋白质复合体和脂类结构域。一组特别耐人寻味的蛋白质是机械力化学酶的Dynamin家族；一个大的GTP酶家族，可能参与几乎所有细胞膜的稳定和分裂事件。我们感兴趣的是研究这些蛋白质的动态结构特性，这些特性来自于它们的机械力化学特性，并将它们与它们的细胞功能相关联。动力蛋白本身对于受体介导的内吞作用、小凹的内化以及高尔基体的来往运输是必不可少的。Dynamin最初与内吞作用有关，当时它被发现是果蝇shibire基因产物的哺乳动物同源物。从那时起，在哺乳动物细胞中过表达的人动力素突变体被发现有效地阻断了笼蛋白介导的内吞作用。我们以前已经证明，纯化的Dynamin很容易组装成环和螺旋，其他人也证明了用GTPgS处理突触体诱导了Dynamin包被的内陷的形成。这一证据支持了这样一种假设，即动力素聚集在覆盖着笼蛋白的凹坑的颈部，在那里它有助于细胞膜的分裂。然而，最近，这一模型变得有争议，出现了另一种建议，认为Dynamin只是作为一个类似于其他GTP酶的分子开关。更多的动力蛋白家族成员参与了许多基本的细胞过程，包括其他膜分裂事件、抗病毒活性、细胞板形成和叶绿体生物发生。在这些蛋白质中，自组装和寡聚为有序结构(即环和螺旋)是一个共同的特征，对大多数蛋白质来说，对它们的功能是必不可少的。虽然它们不断地与细胞的不同功能有关，但我们想知道是否存在共同的作用机制。我们目前正在研究其他Dynamin家族成员，包括参与对抗病毒感染的MxA蛋白和参与线粒体分裂的Dynamin相关蛋白Drp1。 以前，我们证明了Dynamin经历了依赖于GTP的构象变化，导致收缩和碎裂。纯化的重组动力素与脂泡结合形成螺旋管。用GTP处理这些管子会导致结构的快速变化，最终导致收缩和碎裂。我们认为，这代表了当笼蛋白从质膜上萌发时发生的过程中的关键一步。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。为了进一步探索动态蛋白在GTP水解过程中的动力学，我们应用了时间分辨冷冻电子显微镜这一新技术。我们观察到，在加入GTP后(5秒内)，Dynamin立即协同作用收缩潜在的脂双层，多余的脂沿狭窄的管在焦点处凸出。我们目前正在探索不同的动力蛋白突变体、脂质、核苷酸和温度条件来模拟体内环境。为了确定Dynamin诱导收缩过程中发生的构象变化，我们使用冷冻电子显微镜和螺旋重建方法计算了Dynamin在收缩状态下的第一个三维图谱，分辨率为20埃。在GTP类似物存在的情况下，使用缺少富含Pro的C末端(DPRD)的动态蛋白突变体来确定MAP。3D贴图由沿管轴重复的T结构(二聚体)组成，该结构可分为三种不同的密度，称为头部、茎和腿。根据先前的生化结果和X射线晶体结构与我们的图谱的对接，我们预测GTPase结构域位于头部，PH结构域位于腿部。这使得中间结构域和GTP酶效应器域(GED)最有可能位于茎中。GED在茎中的定位与先前的发现相一致，即GED在反式中直接与GTPase结构域相互作用，从而刺激Dynamin的GTPase活性。GTP结合观察到的收缩是由于径向直径和轴向重复数的减少。根据我们的3D图，GED与邻近二聚体的GTPase结构域之间的相互作用可能导致径向和轴向收缩。

The dynamic process of membrane trafficking within eukaryotic cells involves numerous specialized protein complexes and lipid domains. One particularly intriguing set of proteins is the dynamin family of mechanochemical enzymes; a family of large GTPases potentially involved in nearly all cellular membrane stabilization and fission events. We are interested in examining the dynamic structural properties of these proteins, derived from their mechanochemical properties, and correlate them to their cellular function. Dynamin itself is essential for receptor mediated endocytosis, caveolae internalization and trafficking to and from the Golgi. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue to the shibire gene product in Drosophila. Since then human dynamin mutants overexpressed in mammalian cells were found to effectively block clathrin-mediated endocytosis. We have previously shown that purified dynamin readily assembles into rings and spirals, and others have demonstrated that treatment of synaptosomes with GTPgS induced the formation of dynamin-coated invaginations. This evidence supports the hypothesis that dynamin assembles around the necks of clathrin-coated pits where it assists in membrane fission. Recently, however, this model has become controversial and an alternative proposal has emerged suggesting dynamin simply acts as a molecular switch similar to other GTPases. Additional dynamin family members have been implicated in numerous fundamental cellular processes, including other membrane fission events, anti-viral activity, cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures (i.e. rings and spirals) is a common characteristic and, for the majority, essential for their function. While they are continually being implicated in diverse functions of the cell, we would like to know if a common mechanism of action exists. We are currently examining other dynamin family members including the MxA, a protein involved in fighting viral infection and Drp1, a dynamin related protein involved in mitochondria fission. Previously we demonstrated that dynamin undergoes a GTP-dependent conformational change causing constriction and fragmentation. Purified recombinant dynamin binds to lipid vesicles to form helical tubes. Treatment of these tubes with GTP causes a rapid alteration in structure, which ultimately leads to constriction and fragmentation. We believe this represents a critical step in the process that occurs when clathrin-coated pits bud from the plasma membrane. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. To further explore the dynamics of dynamin during GTP hydrolysis we have applied the novel technique of time-resolved cryo-electron microscopy. We observed that immediately upon GTP addition (within 5 seconds) dynamin constricts the underlying lipid bilayer in a concerted action and excess lipid bulges out at focal points along the constricted tubes. We are currently exploring different dynamin mutants, lipid, nucleotide and temperature conditions to mimic the in vivo environment. To determine the conformational changes that occur during dynamin-induced constriction we have calculated the first three-dimensional map of dynamin in the constricted state using cryo-electron microscopy and helical reconstruction methods at a resolution of 20 Angstroms. The map was determined using a dynamin mutant lacking the proline rich C-terminus (DPRD) in the presence of a GTP analogue. The 3D map consists of a repeating T structure (dimer) along the tube axis, which can be divided into three distinct densities called head, stalk and leg. Based on previous biochemical results and the docking of X-ray crystal structures into our map, we predict that the GTPase domain is located in the head and the PH domain is located in the leg. This leaves the middle domain and GTPase effector domain (GED) most likely located in the stalk. The positioning of GED within the stalk fits with previous findings that GED directly interacts in trans with a GTPase domain to stimulate the GTPase activity of dynamin. The constriction observed by GTP binding results from a decrease in both radial diameter and axial repeat. Based on our 3D map, an interaction between GED and a GTPase domain from a neighboring dimer could lead to both a radial and axial constriction.