Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, (19)F NMR, and DFT Approaches

We report X‐ray crystallographic and (19)F NMR studies of the G‐protein RhoA complexed with MgF(3) (−), GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The (19)F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O(3G) and thence to P(G). DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP(Arg85′Ala) relative to wild‐type (WT) RhoGAP. This study demonstrates that (19)F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.