Allosteric role of R97 on the NRas active site

Abstract

Ras proteins are small GTPases that are involved in signal transduction pathways that controls cellular proliferation, differentiation, and survival. Ras is constitutively activated in roughly 20% of human cancers. The Ras subfamily is made up of H‐, K‐, and NRas, where NRas is the least studied. NRas functions as a molecular switch that exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) rendering activation, and hydrolyzes GTP to GDP for deactivation. Once activated, NRas interacts with Raf, an effector molecule that our lab has hypothesized to allosterically modulate intrinsic hydrolysis. The catalytic G domain of NRas comprises an effector lobe (residues 1–86) and allosteric lobe (residues 87–166). In our allosteric model, switch II of Ras becomes ordered when a negatively charged ligand binds between helix 3 and loop 7 allowing for intrinsic hydrolysis. Molecular dynamics studies of Ras bound to Raf revealed R97 of helix 3 becomes less dynamic upon binding of Raf, coordinating the binding of a negatively charged ligand at the allosteric site. Interestingly, R97G is a rare mutation of NRas, found in a human tumor. Here, we present the structure of NRasR97G bound to the non‐hydrolysable GTP analogue, GppNHp. The structure and hydrolysis rates of NRasR97G are compared to those of wild‐type NRas.Support or Funding InformationThis research was funded by NSF Grant Number MCB‐1244203.