Molecular Modeling Reveals the Mechanism of Ran-RanGAP-Catalyzed Guanosine Triphosphate Hydrolysis without an Arginine Finger

Abstract

We report results of a computational study of the reaction mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by the GTP-binding (GTPase) enzyme rat sarcoma-related nuclear (Ran) in complex with its activating protein RanGAP. According to structural investigations, Ran-RanGAP operates without the so-called arginine finger, an arginine residue from a distinct promoter (GTPase-accelerating protein (GAP)) that completes the active site in many GTPases. In this work, we construct model systems for Ran with and without GAP by motifs of the crystal structure of Ran-RanGAP with the GTP analogue and simulate the GTP hydrolysis reaction in these systems. Enzyme–substrate and enzyme–product complexes and reaction intermediates are obtained in quantum mechanics/molecular mechanics (QM/MM) simulations. Calculations of the free-energy reaction profiles are performed at the molecular dynamics level with the ab initio-type QM(DFT(PBE0-D3)/6-31G**)/MM potentials. We show that the computed activation barriers on the pathways for Ran catalysis with and without GAP are in line with the experimentally estimated rate constants. We demonstrate that mapping the Laplacian of the electron density provides easily visible images of substrate activation, which help distinguish between the reactive and nonreactive enzyme–substrate complexes and explain qualitative features of the enzyme-catalyzed GTP hydrolysis reactions consistent with the computed free-energy profiles. A comparison of reactions in Ran and Ran-RanGAP allows us to characterize the role of GAP operating without an arginine finger.