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

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.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase

“…5a are based on reported solution-state NMR assignments 78 , 79 . A similar chemical-exchange process has also been observed for the RhoA/GAP:GDP:AlF 4 − complex 80 , for the GTPase hGBP1 81 and for the motor protein myosin 78 in solution-state 19 F NMR experiments.

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Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase

“…The resonance assignments displayed in Figure 4a are based on reported solution-state NMR assignments 76,77 . A similar chemical-exchange process has also been observed for the RhoA/GAP:GDP:AlF4complex 78 , for the GTPase hGBP1 79 and for the motor protein myosin 76 in solution-state 19 F NMR experiments.…”

Octahedral Trifluoromagnesate, an Anomalous Metal Fluoride Species, Stabilizes the Transition State in a Biological Motor

“… 2 These are tetrahedral BeF 3 – ground state analogues (GSA), octahedral AlF 4 – transition state analogues (TSA) and trigonal bipyramidal (tbp), isosteric MgF 3 – TSA complexes. 2 , 3 …”

“…This partial conversion suggests that for NS3h, the fluoromagnesate TSA is of comparable solution stability to the AlF 4 – TSA. 3 , 14 − 19 …”