Catalytic domain-lipid interactions negatively regulate RhoA signaling by modulating conformational dynamics

RhoA belongs to the Ras superfamily of small GTPases, regulates cytoskeletal actin organization and cell motility, and is implicated in cancer metastasis and neurodegenerative diseases. It is predominantly anchored to the cytosolic side of the plasma membrane (PM) via its prenylated C-terminus, but little is known about its PM organization, dynamics, and lipid interactions. In this work, we used extensive atomistic molecular dynamics (MD) simulations and simulations-guided experiments to study the molecular details and functional consequences of RhoA membrane targeting, interactions and dynamics. We found that both GDP- and GTP-bound RhoA adopt a well-defined, stable membrane organization and binding mode irrespective of initial conditions and bilayer lipid composition. RhoA membrane organization is determined by hydrophobic interaction of the prenylated C-terminus with the membrane, and a previously unknown, critical role of electrostatic interactions between cationic residues on the G-domain and N-terminus with anionic lipids in the PM. N-terminal interactions are mediated by a polybasic patch including residues R5, K6, and K7, and substitution of these residues with Ala increased RhoA-mediated F-actin accumulation and effector activation in cells. MD simulations suggest that the triple mutant retains the global membrane organization of wild type RhoA but alters key protein-lipid interactions. Specifically, we observe a loss of cooperative lipid engagement involving the polybasic patch and Switch II residues with amine-bearing lipids, and a rerouting of intra-protein dynamic networks that primes Switch I for effector recognition. These results suggest that the membrane serves not just as a scaffold but also as an allosteric partner of RhoA to fine-tune intra-protein dynamics in a functionally relevant manner.