High Resolution Structural Biology of the SRP GTPase

描述（由申请人提供）：拟定研究的长期目标是获得对两种GTP酶分子机制的精确功能理解，这两种GTP酶在信号识别颗粒（SRP）介导的分泌蛋白和膜蛋白靶向中发挥核心作用。这两种蛋白质，Ffh，信号序列识别亚基的SRP，和FtsY，其膜相关受体，经历了分子的“握手”的翻译核糖体新生链复合物从胞质SRP转移到膜易位子。值得注意的是，这两种蛋白质的GTdR结构域是结构同源物并直接相互作用。这种瞬时蛋白质：蛋白质复合物的形成机制对于理解根本上重要的SRP靶向机制至关重要。在这里，提出了三条调查路线，以解决其结构基础：第一，在-~ 1.0 A分辨率下确定载脂蛋白和核苷酸结合的Fth GTdR“NG”结构域的结构。完成的结构应允许详细和准确的分析功能上重要的结构元素，没有透露在较低的分辨率确定的结构。第二，Ffh和FtsY的NG结构域的稳定复合物已经在不可水解的GTP类似物的存在下被捕获，并且将进行生物化学表征，目的是使复合物结晶并确定其X射线结构。已经获得了小的“铅”晶体。FfhIFtsY NG复合物的结构将是理解SRP与其受体相互作用的分子细节的基础。最后，将进行生物化学和定点诱变研究，以测试解决两种蛋白质之间相互作用的结构基础的特定假设。这项工作将以前两个目标的成果为基础。未来的研究将致力于了解为什么两个同源的'NG' GTP酶发生在SRP靶向途径的后续步骤。由于SRP GTP酶是一组了解较少的GTP酶的成员，其表现出与经典的“GTP开关”不同的功能逻辑，因此理解形成靶向复合物的结构基础不仅对于SRP，也有助于理解"组装“活化的GTP酶建立在共同的GTP酶折叠上，以利用GTP结合和水解来组织细胞组分以发挥功能。

DESCRIPTION (provided by applicant): The long term objective of the proposed research is to obtain a precise functional understanding of the molecular mechanisms of the two GTPases that play a central role in signal recognition particle (SRP) mediated targeting of secreted and membrane proteins. The two proteins, Ffh, the signal sequence recognition subunit of the SRP, and FtsY, its membrane-associated receptor, undergo a molecular 'handshake' during transfer of the translating ribosome nascent chain complex from the cytosolic SRP to the membrane translocon. Remarkably, the GTPase domains of the two proteins are structural homologs and interact directly. The mechanism for formation of this transient protein:protein complex is central to understanding the fundamentally important SRP targeting mechanism. Here, three lines of investigation to address its structural basis are proposed: First, the structures of the apo- and nucleotide-bound Fth GTPase 'NG' domain are being determined at -~ 1.0 A resolution. The completed structures should allow detailed and accurate analysis of functionally important structural elements that are not revealed in structures determined at lower resolution. Second, the stable complex of the NG domains of Ffh and FtsY has been trapped in the presence of a non-hydrolyzable GTP analog and will be characterized biochemically with the aim of crystallizing the complex and determining its X-ray structure. Small 'lead' crystals have been obtained. The structure of the FfhIFtsY NG complex will be fundamental for understanding the molecular details of the interaction of SRP with its receptor. Finally, biochemical and site-directed mutagenesis studies will be carried out to test specific hypotheses that address the structural basis of the interaction between the two proteins. This work will build on the results of the two previous aims. Future studies will be directed towards understanding why two homologous 'NG' GTPases occur at subsequent steps in the SRP targeting pathway. Because the SRP GTPases are members of a poorly understood group of GTPases that exhibit a functional logic different from the classic 'GTPase switch', an understanding of the structural basis for formation of the targeting complex will be of importance not only with respect to the SRP, but also to understanding how 'assembly-activated' GTPases build on the common GTPase fold to harness GTP binding and hydrolysis to organize cellular components for function.