High Resolution Structural Biology of the SRP GTPase

描述（由申请人提供）：拟议研究的长期目标是获得对两种gtpase的分子机制的精确功能理解，这两种gtpase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。这两个蛋白Ffh （SRP的信号序列识别亚基）和FtsY （SRP的膜相关受体）在翻译核糖体新生链复合物从胞质SRP转移到膜转座子的过程中经历了分子“握手”。值得注意的是，这两种蛋白的GTPase结构域是结构同源的，并直接相互作用。这种瞬时蛋白复合物的形成机制是理解SRP靶向机制的核心。在这里，提出了三条研究路线来解决其结构基础：首先，在-~ 1.0 A分辨率下确定载子和核苷酸结合的第5 GTPase ‘NG’结构域的结构。完成的结构应该允许详细和准确地分析功能重要的结构元素，这些元素在较低分辨率下确定的结构中没有显示出来。其次，Ffh和FtsY的NG结构域的稳定配合物在不可水解的GTP类似物的存在下被捕获，并将进行生物化学表征，目的是结晶该配合物并确定其x射线结构。已经获得了小的“铅”晶体。FfhIFtsY NG复合物的结构将是了解SRP与其受体相互作用的分子细节的基础。最后，将进行生化和定点诱变研究，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。未来的研究将旨在了解为什么两个同源的“NG”gtpase在SRP靶向途径的后续步骤中发生。由于SRP GTPase是一组鲜为人知的GTPase的成员，它们表现出与经典的“GTPase开关”不同的功能逻辑，因此了解目标复合物形成的结构基础不仅对SRP很重要，而且对理解“组装激活”GTPase如何建立在普通GTPase折叠上利用GTP结合和水解来组织细胞成分的功能也很重要。

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.