Ras is a small GTPase involved in the control of cell proliferation and is the focus of this research proposal. Several of its mutants have been directly linked to human cancers, as they remain in an activated state that leads to uncontrolled cell division. Ras is a member of a group of structurally similar proteins involved in highly specific interactions with regulatory agents that control their ability to bind to target proteins within complicated signal transduction cascades. Our objective is to decipher the rules for molecular recognition within the Ras family of GTPases. These rules will be used in the future to guide the design of specific ligands targeted towards the defective oncogenic Ras proteins. Our approach is based on a comparative analysis of the binding surfaces of Ras, two of its most common oncogenic mutants and of its close family members Rap and Ral. Conventional X-ray crystallography will be combined with the multiple solvent crystal structures (MSCS) method where organic solvent molecules are used as probes to the molecular surface features of protein binding sites. Using these methods, we plan to create """"""""functionality maps"""""""" of the binding surfaces of Ras, RasG12V, RasQ61L, Rap and Ral. We will accomplish our goals through four specific aims. First, we will use the MSCS method to characterize both the primary target recognition sites and the putative secondary binding sites on Ras, Rap and Ral, so that the common binding site features across the family can be distinguished from those that are specific to each family member. Second, we will use the MSCS method to determine key differences in the binding site of Ras due to the oncogenic mutations G12V and Q61L. Third, we will solve the crystal structure of Ral and of its complex with the Ral minimum binding domain of its target Ral Binding Protein 1 (RalBP1). We will compare this structure with the analogous complexes for Ras (Ras/RalGDS) and Rap (Rap/Raf), which have previously been published. Fourth, we will combine the results from the first three aims to delineate areas specific to the Ras, and, in particular, to its oncogenic mutants. The goal is to provide key information for ligand design and discovery that is based on multiple structures that offer multiple perspectives. ? ?
|Buhrman, Greg; O'Connor, Casey; Zerbe, Brandon et al. (2011) Analysis of binding site hot spots on the surface of Ras GTPase. J Mol Biol 413:773-89|
|Buhrman, Greg; Kumar, V S Senthil; Cirit, Murat et al. (2011) Allosteric modulation of Ras-GTP is linked to signal transduction through RAF kinase. J Biol Chem 286:3323-31|
|Buhrman, Greg; Holzapfel, Genevieve; Fetics, Susan et al. (2010) Allosteric modulation of Ras positions Q61 for a direct role in catalysis. Proc Natl Acad Sci U S A 107:4931-6|
|Walters, Jad; Pop, Cristina; Scott, Fiona L et al. (2009) A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis. Biochem J 424:335-45|
|Buhrman, Greg; Wink, Glenna; Mattos, Carla (2007) Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf. Structure 15:1618-29|