PROJECT SUMMARY GAPs (GTPase activating proteins) play an essential role in the GTPase cycle, allowing for the regulation of complex processes such as signal transduction; consequently, their dysfunction has significant ramifications. In the case of p120RasGAP (RasGAP, p120; RASA1), the first GAP described, dysfunctions have been linked to vascular diseases such as capillary malformation-arteriovenous malformation syndromes (CM-AVM) and vein of Galen malformations (VOGM). The protein consists of an N-terminal Src homology 2 (SH2)-SH3-SH2 cassette, followed by pleckstrin homology (PH), C2, and GAP domains. Despite its importance and relatively long history as a target of study, p120RasGAP has not been adequately analyzed at the structural and biochemical levels. In particular, although full-length p120RasGAP has been shown to have greater activity than the GAP domain alone, the contributions of the PH and C2 domains have not specifically been isolated. I hypothesize that the GAP activity of p120RasGAP is controlled through conformational changes induced by the protein’s regulatory domains, which are in turn affected by lipid head groups and/or other binding proteins. Specifically, my preliminary data demonstrate that the C2 domain may play a role in GAP regulation. I will test these mechanisms in two aims. In Aim 1, I can already express and purify constructs of the GAP, C2, and PH domains of p120RasGAP, and preliminary enzymatic analysis suggests that the C2 domain accelerates enzymatic activity. I will conduct detailed enzymatic studies in vitro using GAP assays to assess the roles of these domains. I will then assess the effect of disease-causing mutations on enzyme activity. Additionally, I will quantify the affinities of the PH and C2 domains for different lipid head groups and assess C2 calcium sensitivity. These data will then be used to conduct in vitro GAP assays using vesicle-bound Ras to test the role of membrane association in PH and C2 regulatory behavior. In Aim 2, I have already obtained and am refining the first crystal structure of the C2-GAP region of p120RasGAP. This crystal structure demonstrates that the C2 and GAP domains are connected by a flexible linker and illustrates that the C2 domain is ideally positioned to interact with the allosteric lobes of Ras in an “opposable thumb” formation. I will also obtain a crystal structure of the C2- GAP region in complex with HRas to observe this binding directly. Based on analysis of these structures, I will validate interactions made by the C2 domain via site-directed point mutagenesis and GAP assays. Taken together, these aims will provide a comprehensive analysis of the roles of the PH and C2 domains in regulation of p120RasGAP and reveal interactions responsible for the observed changes in activity. These studies will elucidate molecular bases for vascular diseases including CM-AVM and VOGM.