Probing the wild-type HRas activation mechanism using steered molecular dynamics, understanding the energy barrier and role of water in the activation.

Ras is one of the most common oncogenes in human cancers. It belongs to a family of GTPases that functions as binary conformational switches by timely switching of their conformations from GDP to GTP and vice versa. It attains the final active state structure via an intermediate GTP-bound state. The transition between these states is a millisecond-time-scale event. This makes studying this mechanism beyond the scope of classical molecular dynamics. In the present study, we describe the activation pathway of the HRas protein complex along the distance-based reaction coordinate using steered molecular dynamics. Approximately ~720 ns of MD simulations using CMD and SMD was performed. We demonstrated the change in orientation and arrangement of the two switch regions and the role of various hydrogen bonds during the activation process. The weighted histogram analysis method was also performed, and the potential of mean force was calculated between the inactive and active via the intermediate state (state 1) of HRas. The study indicates that water seems to play a crucial role in the activation process and to transfer the HRas protein from its intermediate state to the fully active state. The implications of our study hereby suggest that the HRas activation mechanism is a multistep process. It starts from the inactive state to an intermediate state 1 followed by trapping of water molecules and flipping of the Thr35 residue to form a fully active state (state 2). This state 2 also comprises Gly60, Thr35, GTP, Mg(2+) and water-forming stable interactions.