GTP hydrolysis in RAS

Abstract

RAS GTPases (H, K and NRAS) are closely related membrane associated GTP binding proteins (95% sequence identity) that participate in numerous cell signaling processes, including cellular proliferation and survival. RAS functions as a binary switch, where in the GDP bound form it is signaling inactive, and upon binding GTP it is capable of interacting with effector proteins to propagate the signal. Oncogenic point mutations of RAS are present in 20% of human cancers, and most occur in the GTP binding pocket, affecting the hydrolysis of GTP. A recent hypothesis states that RAS inactivation occurs via an allosterically regulated intrinsic GTP hydrolysis mechanism in the presence of RAF. Despite nearly 25 years of mechanistic proposals, the exact mechanism is still poorly understood due to lack of knowledge of protonation states of the active site residues and of GTP. Neutron crystallography allows for visualization of proton positions in the active site and revealed that RAS is capable of inducing a pKa shift on the γ-phosphate such that it is protonated at pD 8.4, presenting a paradigm shift in our understanding of how the reaction evolves. Additionally, HRAS switch II mutants of the allosteric network were studied using X-ray crystallography and in vitro hydrolysis experiments and the results showed that they are capable of altering the dynamics of the protein as a whole and the catalytic activity of RAS. Furthermore, given that the KRAS isoform is highly implicated in cancer, in vitro hydrolysis experiments were employed for common oncogenic KRAS mutants to assess intrinsic hydrolysis, hydrolysis in the presence of RAF and finally, whether or not a regulatory protein GAP could stimulate them. Results show that they all exhibit significant decreases to the rate compared to wild-type KRAS. The work performed in this thesis provides a foundation upon which future studies can be performed to generate inhibitors, which allosterically target RAS to regulate its oncogenicity.