Structure And Function Of Dynamin, A 100kd GTPase Involv

真核细胞内膜运输的动态过程涉及许多专门的蛋白质复合物和脂质结构域。一组特别有趣的蛋白质是机械化学酶的发动蛋白家族;一个可能参与几乎所有细胞膜稳定和裂变事件的大GTP酶家族。我们感兴趣的是研究这些蛋白质的动态结构特性，来自它们的机械化学性质，并将它们与它们的细胞功能相关联。发动蛋白本身对于受体介导的内吞作用、小窝内化和运输到高尔基体和从高尔基体运输是必需的。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，发动蛋白首次涉及内吞作用。从那时起，在哺乳动物细胞中过表达的人发动蛋白突变体被发现有效地阻断网格蛋白介导的内吞作用。我们以前已经表明，纯化的发动蛋白很容易组装成环和螺旋，和其他人已经证明，与GTPgS处理的突触体诱导形成的发动蛋白包被的内陷。这一证据支持了这样的假设，即发动蛋白聚集在网格蛋白包被的凹坑的颈部周围，在那里它有助于膜分裂。然而，最近，这个模型已经成为有争议的，并出现了一个替代方案，表明发动蛋白只是作为一个类似于其他GTP酶的分子开关。其他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，自组装和寡聚成有序结构（即环和螺旋）是一个共同的特征，并且对于大多数蛋白质来说，对其功能至关重要。虽然它们不断参与细胞的各种功能，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动力蛋白家族成员，包括MxA，一种参与对抗病毒感染的蛋白质和Drp 1，一种参与线粒体分裂的动力蛋白相关蛋白质。 以前，我们证明，发动蛋白经历了GTP依赖的构象变化，导致收缩和碎片。纯化的重组发动蛋白与脂质囊泡结合形成螺旋管。用GTP处理这些管会导致结构的快速改变，最终导致收缩和断裂。我们相信这代表了当网格蛋白包被的小坑从质膜上发芽时发生的过程中的关键步骤。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。为了进一步探索GTP水解过程中发动蛋白的动力学，我们应用了时间分辨冷冻电子显微镜的新技术。我们观察到，GTP加入后立即（5秒内）发动蛋白以协同作用收缩下面的脂质双层，过量的脂质在焦点处沿着收缩的管凸出。我们目前正在探索不同的发动蛋白突变体，脂质，核苷酸和温度条件，以模拟体内环境。为了确定动力蛋白诱导的收缩过程中发生的构象变化，我们已经计算了第一个三维地图的动力蛋白在收缩状态下使用冷冻电子显微镜和螺旋重建方法在20埃的分辨率。使用缺乏富含脯氨酸的C-末端（DPRD）的发动蛋白突变体在GTP类似物的存在下确定该图。3D图由沿管轴沿着的重复T结构（二聚体）组成，其可分为三种不同的密度，称为头、柄和腿。根据以前的生化结果和X射线晶体结构的对接到我们的地图，我们预测，GTdR域位于头部和PH域位于腿部。这使得中间结构域和GTP酶效应子结构域（GED）最可能位于茎中。GED在茎内的定位符合先前的发现，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。GTP结合所观察到的收缩是由径向直径和轴向重复减少引起的。基于我们的3D图，GED和来自相邻二聚体的GTdR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.