Conformational Transitions in Switch Regions of the Ras-Like GTPase Rab1B Studied by Free Energy Simulations

Abstract

The family of small Ras-like GTPases plays a pivotal role in the coordination of cell signaling by acting as binary protein switches. The switching mechanism is triggered by the hydrolysis of co-factor GTP to GDP which induces a transition in conformation and dynamics of two functional regions designated as switch I and switch II located in the proximity of the co-factor binding site. The switch I and switch II regions typically adopt an ordered and well defined structure only in the presence of bound GTP and unfold in the presence of GDP. The conformational equilibrium of switch I and switch II regions can be further influenced by post-translational modifications. We employed umbrella sampling free energy simulations to systematically investigate the switch I and switch II conformational transitions in the Ras-like GTPase Rab1b in the presence of bound GTP or GDP and different posttranslational modifications. The free energy change was recorded along a collective coordinate composed of a network of predefined inter-atomic distances (dRMSD) coupled with Hamiltonian replica exchange simulations. In agreement with experiment the free energy simulations predicted a lower free energy barrier of unfolding switch I and switch II regions in the presence of GDP compared to GTP and are also compatible with the experimentally observed effects of posttranslational modifications. An energetic analysis indicates that electrostatic interactions play a decisive role for the conformational equilibrium. It was also possible to determine the order of structural transitions in the switch I and II regions upon induced unfolding. The results can have important implications for understanding the mechanism of signal transduction by GTPases.