STRUCTURAL STUDY OF RHO-GTPASE REGULATORS AND EFFECTORS

Rho家族GTPases Cdc 42、Rac和Rho调节细胞骨架 结构、粘附和细胞中的基因表达。 异常信号传导 通过这些蛋白质可以导致细胞转化和肿瘤 侵略性 本报告的研究重点是比较 通过NMR光谱对两种配体进行结构表征， Cdc 42 Hs GT3、负调节因子、RhoGDI和CRIB结构域 Wiskott-Aldrich综合征蛋白（WASP）。 RhoGDI的初步研究表明，该蛋白质由两个 具有不同但可能重叠的功能的不同区域。 N-末端结构域结合Cdc 42 Hs的蛋白质部分，而 C-末端结构域结合异戊二烯基。的骨干和 侧链1H，15 N和13 C化学位移分配的16 kDa C- RhoGDI末端片段（GDIC，残基60-204）几乎 完成. 该结构域的结构将被确定为高 分辨率使用NOE和3 J耦合信息来自3-和 4-三维NMR实验。 GDIC络合物的结构 其中异戊二烯化肽代表Cdc 42 H的C-末端 也将被确定。 这些结构可以解释 RhoGDI及其同源物通过其从膜提取GTP酶， 从而负调控Rho家族信号传导。 n-末端 RhoGDI结构域（GDIN）将在与Cdc 43 Hs的复合物中进行研究 通过一系列的实验， 信息的分辨率越来越高。 GDIN结合 将绘制GTcycle的表面，然后绘制结构 确定约束调节器，并最终确定 复杂. 该系统的相对大的尺寸（26 kDa）将需要 扩展最近开发的氘辅助NMR方法， 多蛋白质复合物的分析。 结构和生化 对RhoGDI结构域的分析将解释蛋白质结合 所有Rho-GTP酶，抑制核苷酸的解离，并阻断 其他调节分子的作用。 WASP CRIB的复杂性 结构域和Cdc 42 H也将以类似的方式进行研究，利用 新的氘辅助NMR方法。 结构数据比较 RhoGDI和WASP将解释 这两种配体，包括它们对GT3的需求 异戊二烯化、对核苷酸状态的敏感性和对 不同的罗家族成员 这些研究将提供基本的 深入了解通过Rho信号转导的机制， GTP酶，并将开辟新的途径，为发展诊断和 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.