A GAP‐GTPase‐GDP‐P_i Intermediate Crystal Structure Analyzed by DFT Shows GTP Hydrolysis Involves Serial Proton Transfers

Abstract

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.

Common patterns of hydrolysis initiation in P-loop fold nucleoside triphosphatases

“…1D, 7C). Reorientation of the Wcat molecule into the attack position and its polarization, as suggested by Jin and colleagues [44,64,65], can be realized by those Arg or Lys fingers that reach Wcat (Fig. 6D), but not by most other stimulators.…”

“…23.497298 doi: bioRxiv preprint bound water molecules out of the binding pocket into the bulk solution by the stimulatory arginine finger provides an entropic gain [63]. Jin and colleagues proposed reorientation of the Wcat molecule into the attack position by the stimulator [44,64,65]. On a later reaction step, stimulators were implied in compensating for the negative charge that develops at β-phosphate upon the breakaway of γ-phosphate [49,66].…”

Common mechanism of activated catalysis in P-loop fold nucleoside triphosphatases -in varietate concordia

“…According to the model, the catalytic transition proceeds in the following steps: E) The proton that comes from [Ser/Thr] K+1 forms a new short H-bond between β-and γphosphate [52,201,281], which further stabilizes the negative charge on the β-phosphate. The proton at Asp WB relocates to [Ser/Thr] K+1 .…”

Recent Advances in Understanding Biological GTP Hydrolysis through Molecular Simulation

“…We note that the proton shuttling proposed in ref is based on the assumption of a substrate-assisted pathway (Figure C), in which the attacking water molecule in the Rho active site is initially deprotonated by the bound nucleotide. Similar assumptions of mobile protons have been put forward for Ras and hGBP1, , in the former case involving the active site glutamine shown in Figure , and in the latter case a more complex proton transfer chain involving multiple active site side chains and an intermediary water molecule.…”

“…They also provided novel spectra of GTP and several GTP analogues bound to lipidated Ras, on a membrane system under near-native conditions, confirming that the nucleotide is indeed bound to Ras in its fully deprotonated form at the start of the hydrolysis reaction and is not protonated. In detailed crystallographic and quantum chemical analysis of an analogous system, a quaternary complex of Rho• GAP•GDP•P i , 21 the authors argue for a complex network of hydrogen bonds and sequential proton transfers, including two low-barrier hydrogen bonds (LBHB) introduced by the GAP, that facilitate reaction progress and product release. We note that the proton shuttling proposed in ref 21 is based on the assumption of a substrate-assisted pathway (Figure 1C), in which the attacking water molecule in the Rho active site is initially deprotonated by the bound nucleotide.…”