STRUCTURAL STUDY OF RHO-GTPASE REGULATORS AND EFFECTORS

Rho家族GTP酶CDc42、Rac和Rho调节细胞骨架 细胞的结构、黏附和基因表达。异常信号 通过这些蛋白质可以导致细胞转化和肿瘤 侵犯性。本提案中的研究侧重于比较 两种配体的核磁共振波谱结构表征 CDC42Hs GTP酶、负性调节因子、RhoGDI和KRIB结构域 可能的效应蛋白是Wiskott-Aldrich综合征蛋白(WASP)。 RhoGDI的初步研究表明，该蛋白由两个 不同的区域具有不同的但可能重叠的功能。 N-末端结构域结合CDC42Hs的蛋白质部分，而 C-末端结构域与异戊二烯基团结合。主心骨和 16 kDa C-侧链的1H、15N和13C化学位移归属 RhoGDI的末端片段(GDIC，残基60-204)几乎 完成。这个域的结构将被确定为高 使用NOE和3J耦合信息的分辨率从3-和 四维核磁共振实验。GDIC络合物的结构 用异戊二烯基化的多肽表示Cdc42Hs的C-末端 也将被确定。这些结构将解释这种机制。 RhoGDI及其同系物通过其从膜中提取GTP酶， 因此，负调控Rho家族信号。N端子 RhoGDI(GDIN)的结构域将与Cdc43Hs形成络合物进行研究 通过一系列旨在提供结构的实验 在不断提高的分辨率水平上的信息。GDIN结合 将绘制GTP酶的表面，然后是结构 确定受约束的调节器，并最终确定全部 很复杂。该系统相对较大的尺寸(26 KDa)将需要 最新发展的氚辅助核磁共振方法的扩展到 多蛋白质复合体的分析。结构和生化 对RhoGDI结构域的分析将解释蛋白质的结合能力 所有的Rho-GTP酶，抑制核苷酸的解离，并阻断 其他调节分子的作用。WASP婴儿床的情结 结构域和Cdc42H也将以类似的方式进行研究，利用 新的氢辅助核磁共振方法。的结构数据比较 RhoGDI和WASP将解释不同的绑定属性 这两种配体及其对GTP酶的要求 异丙二烯基化，对核苷酸状态的敏感性，以及对 不同的Rho家族成员。这些研究将提供基本的 对Rho信号转导机制的见解-- GTP酶，并将开辟新的途径，发展诊断和 作用于这些途径的治疗剂。

The Rho-family GTPases Cdc42, Rac and Rho regulate cytoskeletal structure, adhesion and gene expression in cells. Aberrant signaling through these proteins can lead to cellular transformation and tumor invasiveness. Research in this proposal focuses on comparative structural characterization by NMR spectroscopy of two ligands for the Cdc42Hs GTPase, the negative regulator, RhoGDI and CRIB domain of the putative effector, Wiskott-Aldrich Syndrome protein (WASP). Preliminary studies of RhoGDI indicate the protein is composed of two distinct regions with different, but possibly overlapping functions. The N-terminal domain binds the protein portion of Cdc42Hs, while the C-terminal domain binds to isoprenyl groups. The backbone and sidechain 1H, 15N and 13C chemical shift assignments of a 16 kDa C- terminal fragment of RhoGDI (GDIC, residues 60-204) are nearly complete. The structure of this domain will be determined to high resolution using NOE and 3J coupling information derived from 3- and 4-dimensional NMR experiments. The structure of a complex of GDIC with an isoprenylated peptide representing the C-terminus of Cdc42Hs will also be determined. These structures will explain the mechanism by which RhoGDI and its homologs extract GTPases from membranes, thus negatively regulation Rho-family signaling. The N-terminal domain of RhoGDI (GDIN) will be studied in complex with Cdc43Hs through a series of experiments designed to provide structural information at increasing levels of resolution. The GDIN-binding surface of the GTPase will be mapped, followed by structure determination of the bound regulator, and ultimately of the full complex. The relatively large size of this system (26kDa) will require extension of recently developed deuterium-aided NMR methods to analysis of multi-protein complexes. Structural and biochemical analyses of RhoGDI domains will explain the protein's ability to bind all Rho-GTPases, inhibit dissociation of nucleotides, and block the actions of other regulatory molecules. The complex of the WASP CRIB domain and Cdc42Hs will also be studied in a similar manner, utilizing novel deuterium-aided NMR methods. Comparison of structural data on RhoGDI and WASP will explain the different binding properties of these two ligands, including their requirements for GTPase isoprenylation, sensitivity to nucleotide state, and specificity for different Rho-family members. These studies will provide fundamental insights into the mechanisms of signal transduction through Rho- GTPases, and will open new avenues for development of diagnostic and therapeutic agents that act on these pathways.