Classification of GTP-dependent K-Ras4B active and inactive conformational states

Classifying reliably active and inactive molecular conformations of wildtype and mutated oncogenic proteins is a key, ongoing challenge in molecular cancer studies. Here, we probe the GTP-bound K-Ras4B conformational dynamics using long-time atomistic molecular dynamics (MD) simulations. We extract and analyze the detailed underlying free energy landscape of wildtype (WT) K-Ras4B. We use two key reaction coordinates, labelled d1 and d2 (i.e., distances coordinating the Pb atom of the GTP ligand with two key residues, T35 and G60), shown to correlate closely with activities of WT and mutated K-Ras4B. However, our new K-Ras4B conformational landscape reveals a more complex network of equilibrium Markovian states. We show that a new reaction coordinate is required to account for the orientation of acidic K-Ras4B sidechains such as D38 with respect to the interface with binding effectors such as RAF1 and is needed to rationalize the activation/inactivation propensities and the corresponding molecular binding mechanism. We use this understanding to unveil how a relatively conservative mutation (i.e., D33E, in the switch I region) can lead to significantly different activation propensities compared to WT K-Ras4B. Our study sheds new light on the ability of residues near the K-Ras4B - RAF1 interface to modulate the network of salt bridges at the binding interface with the RAF1 downstream effector and, thus, to influence the underlying GTP-dependent activation/inactivation mechanism. Altogether, our hybrid MD-docking modelling approach enables the development of new in silico methods for quantitative assessment of activation propensity changes and facilitates the rational design of new cancer drugs.