Molecular mechanism of GTPase-activating protein-assisted hydrolysis of GTP catalyzed by Cdc42.

Cell division control protein 42 homolog (Cdc42), a key regulator of cell proliferation and migration implicated in cancer progression, cycles between active GTP-bound and inactive GDP-bound states. GTPase-activating proteins (GAPs) promote Cdc42 inactivation by accelerating GTP hydrolysis, but the underlying catalytic mechanism remains incompletely understood. Here, integrated molecular dynamics (MD) simulations and density functional theory (DFT) calculations demonstrate that GAP binding promotes hydrolysis through a combination of active-site preorganization, enhanced electrostatic interactions with phosphate, and Arg305-assisted stabilization of the Gln61 position and electrostatics. The Gln61-assisted proton transfer pathway, where Gln61 aligns the water molecule for nucleophilic attack and facilitates proton transfer, is energetically favored over direct proton transfer within the selected preorganized MD conformations. GAP binding is associated with a lower hydrolysis barrier, consistent with GAP-assisted Cdc42 inactivation. These findings elucidate the structural dynamics of the Cdc42-GAP complex and the catalytic role of GAP in GTP hydrolysis. This mechanistic framework can inform future studies of Cdc42 regulation and its relevance to cancer-related processes.