Structure and catalytic properties of NRas and its oncogenic mutants

Abstract

Ras GTPases are signaling proteins that exist in three isoforms, N-Ras, K-Ras and H-Ras that are involved in a variety of signal transduction pathways, including cellular proliferation and survival. Ras acts as a molecular switch, where it is inactive when bound to GDP and propagates its signal when bound to GTP. The three Ras isoforms have identical effector lobe (residues 1-86 containing the active site and effector binding regions), 90% conserved allosteric lobe (residues 87-166) with influence on active site conformational states, and a C-terminal 15% conserved hypervariable region (residues 167-189). In Ras bound to GTP analogues, differences in allosteric lobe stabilize conformational states with distinct biochemical properties, including rate constants for GTP hydrolysis and affinity for effector proteins. When bound to GTP, switch I in the active site interconverts between an open conformation (state I) that favors nucleotide exchange and a closed conformation (state 2) associated with effector binding and GTP hydrolysis. Little efforts have been made to elucidate biochemical differences among the three isoforms of Ras. Earlier works underscored kinetic differences between HRas and NRas, however, these studies were ignored due to the conserved active sites in the isoforms. NRas is frequently mutated in melanoma tissues at Q61, however there are currently no approved inhibitors that target NRas. Targeting NRas has been stunted due to the limited structural knowledge of NRas and its mutants in the literature. The work presented here elucidates the structural and biochemical properties of NRas and its oncogenic mutants. The first crystal structure of NRas bound to a GTP analogue is presented that support the notion that the three Ras isoforms are distinct. Further we assess the balance of conformational states of NRasQ61X mutants using kinetics, NMR data, and molecular dynamic simulations. This work provides insight that can be used in developing therapeutic strategies against NRas mutant cancers.