NMR STRUCTURAL STUDIES OF THE GTPASE RAC1

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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.