Rho GTPases are members of the Ras superfamily of small GTPases that regulate cell morphology, motility, polarization and cell cycling. Like members of the Ras subfamily, Rho subfamily GTPases dysregulation is implicated in a range of tumors and can serve as a valid drug target. In this work, we investigate the evolutionary trajectory of Rho GTPases within a region of the protein that has been exploited for cancer drug discovery within the Ras subfamily branch - the "switch II pocket". Our previous work has illustrated the role of allostery in this region of H-Ras in modulation of intrinsic hydrolysis and effector-binding capacity. Here, we report that a highly conserved salt bridge within the Rho subfamily stabilizes the RhoA GTPase active site in a catalytically favorable conformation. We probed the roles of the Rho salt bridge via X-ray crystallography, accelerated molecular dynamics simulations (aMD), and enzymatic studies. We showed that the removal of a residue within switch II of RhoA, the salt bridge residue R70, can impart catastrophic effects on active site organization and GTP hydrolysis. As expected, removal of the analogous R68 in H-Ras, which is not involved in a salt bridge interaction, results in a structure with changes in the active site and a decrease in GTP hydrolysis rate constant that are more moderate than observed for RhoA. The anionic partner of R70, E102, also modulates active site conformation and, upon removal, decreases intrinsic hydrolysis. Based on aMD simulations, we uncovered evidence of epistatic relationships between the Rho salt bridge, the distal residue K98 and P-loop residue D13 which coordinate allosteric communication from the switch regions directly to the active site. Finally, we describe the functional landscape of switch II pocket in the context of both Rho subfamily evolution and potential for drug discovery.
Marcus et al. (2026) studied this question.