Browsing by Author "Pellegrini, Erika"

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    Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches
    (Wiley, 2017-08-07) Jin, Yi; Molt, Robert W.; Pellegrini, Erika; Cliff, Matthew J.; Bowler, Matthew W.; Richards, Nigel G. J.; Blackburn, G. Michael; Waltho, Jonathan P.; Biochemistry and Molecular Biology, School of Medicine
    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.
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    A GAP‐GTPase‐GDP‐Pi Intermediate Crystal Structure Analyzed by DFT Shows GTP Hydrolysis Involves Serial Proton Transfers
    (Wiley, 2019) Jin, Yi; Molt, Robert W., Jr.; Pellegrini, Erika; Biochemistry and Molecular Biology, School of Medicine
    Cell signaling by small G proteins uses an ON to OFF signal based on conformational changes following the hydrolysis of GTP to GDP and release of dihydrogen phosphate (Pi). The catalytic mechanism of GTP hydrolysis by RhoA is strongly accelerated by a GAP protein and is now well defined, but timing of inorganic phosphate release and signal change remains unresolved. We have generated a quaternary complex for RhoA‐GAP‐GDP‐Pi. Its 1.75 Å crystal structure shows geometry for ionic and hydrogen bond coordination of GDP and Pi in an intermediate state. It enables the selection of a QM core for DFT exploration of a 20 H‐bonded network. This identifies serial locations of the two mobile protons from the original nucleophilic water molecule, showing how they move in three rational steps to form a stable quaternary complex. It also suggests how two additional proton transfer steps can facilitate Pi release.