Assessing the Influence of Mutation on GTPase Transition States using X-ray, 19F NMR, and DFT Approaches

We report X-ray crystallographic and 19F NMR studies of
RhoA complexed with MgF3
–, GDP, and RhoGAP having the
mutation Arg85’Ala, which, when combined with DFT calculations,
permit the identification of changes in TS properties. Thus, X-ray
shows how Tyr34 maintains solvent exclusion and the core H-bond
network in the active site by relocating to replace the missing Arg85’
sidechain. 19F 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 O3G and
thence to PG. DFT calculations identify electron density redistribution
and pinpoint why the TS for GTP hydrolysis is higher in energy when
RhoA is complexed with RhoGAPArg85’Ala relative to WT RhoGAP.
This study demonstrates that 19F NMR measurements, in
combination with X-ray and DFT, can reliably dissect the response of
small GTPases to site-specific modification.