Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches. Issue 33 (24th May 2017)

• Record Type: Journal Article
• Authors: Jin, Yi; Molt, Robert W.; Pellegrini, Erika; Cliff, Matthew J.; Bowler, Matthew W.; Richards, Nigel G. J.; Blackburn, G. Michael; Waltho, Jonathan P.
• Is Part Of: Angewandte Chemie international edition. Volume 56:Issue 33(2017)
• Page Start: 9732
• Page End: 9735
• Publication Date: 2017-05-24
• Subjects: 19F NMR -- DFT calculations -- enzyme mechanisms -- GTPases -- RhoA/RhoGAP; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201703074
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: We report X‐ray crystallographic and 19 F 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 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 RhoGAPArg85′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. Abstract : In transit : Mutagenic removal of an arginine finger from the GTPase‐activating protein RhoGAP leads to changed crystal structures and transposed 19 F NMR data for MgF x transition‐state analogue (TSA) complexes, owing to the exclusively electrostatic contribution of Arg85′. This was fully delineated through DFT computation of electron distribution in the transition state. Taken together the data show a later, more dissociative transition state for phosphoryl transfer.