Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches
Year: 2017
Type: article
Abstract: We report X‐ray crystallographic and 19F NMR studies of the G‐protein RhoA complexed with MgF3−, 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 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 guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that 19F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $2,721
Awards: The influence of metal fluorides on the structure and dynamics of phosphoryl transfer enzymes, Dynamics, Gating and Opening in Enzyme Catalysis, Understanding enzyme-catalysed phosphoryl transfer, G4202253467, Enzyme catalysis of nucleophilic attack of anions by anions, G590879898, G6497890396, G835584914
OpenAlex ID: W2613300067