Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches
Open Access
- 24 May 2017
- journal article
- research article
- Published by Wiley in Angewandte Chemie International Edition
- Vol. 56 (33) , 9732-9735
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.Keywords
Funding Information
- Biotechnology and Biological Sciences Research Council (BB/M021637/1, BB/E017541/1, BB/K016245/1)
This publication has 30 references indexed in Scilit:
- Regulation of Small GTPases by GEFs, GAPs, and GDIsPhysiological Reviews, 2013
- Charge‐Balanced Metal Fluoride Complexes for Protein Kinase A with Adenosine Diphosphate and Substrate Peptide SP20Angewandte Chemie, 2012
- Charge‐Balanced Metal Fluoride Complexes for Protein Kinase A with Adenosine Diphosphate and Substrate Peptide SP20Angewandte Chemie International Edition, 2012
- Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulationsProceedings of the National Academy of Sciences, 2012
- Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrateProceedings of the National Academy of Sciences, 2012
- Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and CatalysisAnnual Review of Biochemistry, 2011
- Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranesProceedings of the National Academy of Sciences, 2010
- A Trojan horse transition state analogue generated by MgF 3 − formation in an enzyme active siteProceedings of the National Academy of Sciences, 2006
- Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAPNature Structural & Molecular Biology, 1998
- GTPase mechanism of Gproteins from the 1.7-Å crystal structure of transducin α - GDP AIF−4Nature, 1994