Structure and Function of the Dbl Homology Domain of Human Beta-PIX, a Guanine Nucleotide Exchange Factor for the Rho GTPases

RosenMCB 98173761。Rho家族中的algt帕斯酶调节细胞骨架结构、粘附、运动和基因表达。rho家族信号传导的一个关键尚未解决的问题是gtpase如何被dbl家族中的上游鸟嘌呤核苷酸交换因子（gef）激活。本提案描述了最近发现的db -家族GEF， human PIXB （hPIXB）的结构和功能分析。工作的初始阶段将集中在hpixb的Dbl-homology （DH）域的核磁共振结构确定。结构域的大尺寸和较差的脂肪族化学位移分散将需要使用选择性甲基和芳香标记策略来获得侧链化学位移分配和结构域的全局折叠。这项工作将代表这些策略首次应用于未知折叠的蛋白质，因此将为它们在核磁共振结构测定中更大蛋白质的实用性提供关键测试。该结构将促进整个Dbl家族的氨基酸序列分析，为dhdomain的催化机制和GTPase识别提供重要线索。该结构以及GTPase界面的核磁共振图谱将指导诱变实验，以确定特定氨基酸在介导GEF催化中的作用。与其他GEF结构域的结构和功能数据的比较将揭示核苷酸交换催化的一般原理。最后，在所有DH结构域的c端发现的plekstrin同源结构域（PH）结构域在调节gtpase结合和GEF活性中的作用将进行生化和光谱学研究。这些结果将解释hPIXB PH结构域是否仅仅作为膜靶向序列或作为DH结构域GEF活性的变构调节剂。这一信息将对细胞中DH蛋白的调控和gtpase的激活机制产生影响。Rho gtp酶代表了一类重要的蛋白质，它们传递控制细胞形状、粘附和运动的信号。这些分子在不活跃的gdp结合状态和活跃的gtp结合状态之间循环。当与GTP（而不是GDP）络合时，它们对选定的靶蛋白具有很强的亲和力，并且与这些分子结合构成细胞内的信息传递。鸟嘌呤核苷酸交换因子（GEFs）属于Dbl家族，是一种催化gdp释放和GTP再结合的酶，从而增加活化Rho蛋白的细胞数量。本研究的重点是了解Dbl蛋白对Rho gtpase活性的分子机制。最初的工作是利用核磁共振波谱（NMR）测定一个Dblfamily成员hPIXB的三维结构。除了提供重要的功能信息外，这些研究还将为核磁共振结构测定新策略的开发提供平台。这项工作将随后进行hPIXB突变体的生化研究，这将使鉴定蛋白质中对活性重要的单个氨基酸成为可能。最后，有证据表明，Dbl蛋白的活性可能通过催化结构域与相邻调节模块的相互作用以分子内方式进行调节。随后将进行光谱和生化分析，以探究这种相互作用的性质和功能。总之，这些研究将解释用于调节细胞中活化的Rho家族gtpase水平的许多生化和结构机制。

RosenMCB 98173761. TechnicalGTPases in the Rho family regulate cytoskeletal structure,adhesion, motility, and gene expression in cells. A key unresolved issuein Rho-family signaling is how the GTPases are activated by upstreamguanine nucleotide exchange factors (GEFs) in the Dbl-family. Thisproposal describes structural and functional analysis of a recentlydiscovered Dbl-family GEF, human PIXB (hPIXB). Initial stages of the workwill focus on structure determination of the Dbl-homology (DH) domain ofhPIXB by NMR spectroscopy. The large size and poor aliphatic chemicalshift dispersion of the domain will necessitate use of selective methylgroup and aromatic labeling strategies to obtain sidechain chemical shiftassignments and a global fold of the domain. The work will represent thefirst application of these strategies to a protein of unknown fold, and assuch will provide a critical test of their utility in structuredetermination of larger proteins by NMR. The structure will facilitateamino acid sequence analyses across the Dbl family, providing importantclues regarding mechanisms of catalysis and GTPase recognition by DHdomains. The structure, along with NMR mapping of the GTPase interface,will then guide mutagenesis experiments to identify the roles of specificamino acids in mediating GEF catalysis. Comparisons with structural andfunctional data on other GEF domains will then reveal general principles ofnucleotide exchange catalysis. Finally, the role of the plekstrin homology(PH) domain, found immediately C-terminal to all DH domains, in regulatingGTPase binding and GEF activity will be examined biochemically andspectroscopically. The results will explain whether the hPIXB PH domainfunctions simply as a membrane-targeting sequence or as an allostericregulator of DH domain GEF activity. This information will haveimplications regarding the regulation of DH proteins and mechanisms ofGTPase activation in the cell.2. Non-technicalThe Rho GTPases represent an important class of proteins thattransmit signals controlling cell shape, adhesion and movement. Thesemolecules cycle between an inactive GDP-bound state and an active GTP-boundstate. When complexed with GTP, but not GDP, they have strong affinity forselected target proteins, and binding to these molecules constitutestransmission of information within the cell. Guanine nucleotide exchangefactors (GEFs) in the Dbl family are enzymes that catalyze release of GDPand rebinding of GTP, thus serving to increase the cellular population ofactivated Rho proteins. This study focuses on understanding the molecularmechanisms of Dbl protein activity toward the Rho GTPases. Initial workdeals with determination of the three-dimensional structure of one Dblfamily member, hPIXB, using nuclear magnetic resonance spectroscopy (NMR).In addition to providing important functional information, these studieswill also provide a platform for development of new strategies forstructure determination by NMR. This work will be followed by biochemicalstudies of hPIXB mutants that will enable identification of individualamino acids in the protein that are important for activity. Finally, thereis evidence that the activities of the Dbl proteins may be regulated in anintramolecular fashion through interactions of the catalytic domain with anadjacent regulatory module. Spectroscopic and biochemical assays will besued to probe the nature and function of this interaction. Together, thesestudies will explain many of the biochemical and structural mechanisms usedto regulate the level of activated Rho family GTPases in the cell.