NMR STRUCTURAL STUDIES OF THE GTPASE RAC1

Rac1是一种小型鸟苷三磷酸酶（GTPase），在肌动蛋白在质膜上的聚合以及板足和膜褶的形成中起重要作用。Rac1属于gtpase的Ras超家族，并保留了与鸟嘌呤核苷酸结合有关的一致残基。然而，Rac1与Ras只有30%的序列同源性，并且在功能上不同，参与多种细胞过程，包括细胞凋亡和基因表达。与Ras不同，Rac1还具有一个13个氨基酸的插入，据信在蛋白质-蛋白质相互作用中起作用。最近发现的一种失活性突变形式的Rac1的晶体结构表明，Rac1具有与Ras相似的拓扑结构，但在蛋白质的重要功能区域与Ras不同。目前尚不清楚这些差异是由于突变、晶体填塞力，还是代表两种蛋白质之间的真正差异。因此，我们研究的主要目标将是通过高分辨率核磁共振波谱技术对Rac1的GTP-和gdp -形式进行结构表征。我们设计了许多在插入区和羧基末端有突变/缺失的Rac1变异，以了解插入和羧基末端在Rac1介导的细胞信号传导中的作用。这些区域在gtpase的Rho家族中是非常独特的，并且在Rho gtase -蛋白相互作用中起重要作用。我们已经启动了对这些Rac1变异的结构、生化和生物学研究，以阐明Rac1如何通过多种信号传导途径转导信号并促进多种生物反应。细胞生物学研究是与北卡罗来纳大学钱宁·德博士的实验室合作进行的。我们对野生型和突变型RTac1的研究将为了解Rac1的分子、生化和生物学特征提供重要信息，这些特征对Rac1 GTP/GDP循环至关重要，并在Rac1介导的细胞信号传导中发挥重要作用。本课题的具体目的是：1)确定全长Rac1与GMPPNP络合的核磁共振溶液结构；3)表征Rac1突变体的生物学和生化特性；4)通过核磁共振光谱对突变体Rac1蛋白进行结构表征。我们研究的最终目标是阐明GTPase Rac1的结构/功能关系，并描述该蛋白如何将GDP-GTP交换偶联到体内下游靶标的激活。这些研究也将为我们的长期目标奠定基础，即研究调节Rac功能的药物的结合模式。这些研究应该为癌症的治疗干预提供有用的信息。

This proposal is focused on Rac1, a small guanosine triphosphatase (GTPase) that plays an important role in actin polymerization at the plasma membrane and in the formation of lamellipodia and membrane ruffles. Rac1 belongs to the Ras superfamily of GTPases and retains the consensus residues that are involved in binding guanine nucleotides. However, Rac1 shares only 30% sequence identity with Ras and is functionally distinct being involved in a wide variety of cellular processes which include apoptosis and gene expression. Unlike Ras, Rac1 also possesses a 13-amino acid insert that is believed to play a role in protein- protein interactions. The crystal structure of an inactive mutant form of Rac1 solved recently shows that Rac1 has a similar topology as Ras but differs from Ras in functionally important regions of the protein. It is not clear whether these differences are due to mutation, crystal packing forces, or represent true differences between the two proteins. Therefore the main goal of our studies will be to structurally characterize the GTP- and GDP-forms of Rac1 by high resolution NMR spectroscopy. We have designed a number of Rac1 variants with mutations/deletions in the insert region and the carboxyl-terminus to understand the roles of the insert and the carboxyl-terminus in Rac1 mediated cell signaling. These regions are very distinct in the Rho family of GTPases and have been 8implicated to play an important role in Rho GTPase-protein interaction. We have initiated structural, biochemical and biological studies on these Rac1 variants to elucidate how Rac1 transduces signals through multiple signaling pathways and contributes to a diverse array of biological responses. The cell biology studies are being conducted in collaboration with the laboratory of Dr. Channing Der at the University of North Carolina. Our proposed studies of wild type and mutant forms of RTac1 will provide important information for understand the molecular, biochemical and biological features of Rac1 critical for Rac1 GTP/GDP cycling and its importance in Rac1-mediated cell signaling. The specific aims of this proposal are to: 1) To determine the NMR solution structure of full length Rac1 complexed to GMPPNP 3) To characterize the biological and biochemical properties of Rac1 mutants 4) To structurally characterize mutant Rac1 proteins by NMR spectroscopy. The ultimate goal of our studies is to elucidate structure/function relationships in the GTPase Rac1 and to delineate how this protein couples GDP-GTP exchange to the activation of downstream targets in vivo. These studies will also lay the groundwork for our longer term goals, which are to investigate the binding mode of agents that modulate Rac function. Such studies should provide information helpful for therapeutic intervention in cancer.