Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate

Knihtila, Ryan; Holzapfel, Genevieve; Weiss, Kevin L.; Meilleur, Flora; Mattos, Carla

doi:10.1074/jbc.m115.679860

Cited by 45 publications

(55 citation statements)

References 58 publications

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“…Finally, another issue relates to the protonation state of the γ phosphate in Ras‐GTP, which is thought to be fully deprotonated based on the pH dependence of P NMR chemical shifts . In a recent neutron scattering structure of ground state Ras‐GppNHp exchanged with D 2 O, however, a deuteron is observed on one of the γ phosphate oxygens at a calculated pD of 8.4 . This apparent discrepancy may be due to differences in the pK a of the γ phosphate, which is higher for GppNHp than GTP in solution Given the potential mechanistic implications, the question of whether the γ phosphate is protonated in Ras‐GTP with or without RasGAP would benefit from clarification.…”

Section: Small Gtpase Architecturementioning

confidence: 99%

“…52,86,89 In a recent neutron scattering structure of ground state Ras-GppNHp exchanged with D 2 O, however, a deuteron is observed on one of the c phosphate oxygens at a calculated pD of 8.4. 90 This apparent discrepancy may be due to differences in the pK a of the c phosphate, which is higher for GppNHp than GTP in solution 52 Given the potential mechanistic implications, the question of whether the c phosphate is protonated in Ras-GTP with or without RasGAP would benefit from clarification. It might be interesting, for example, to compare the protonation of state of constitutively active Ras mutants loaded with GTP vs. GppNHp.…”

Section: Chemical Kinetic Barriers and Reaction Pathwaysmentioning

confidence: 99%

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Invited review: Small GTPases and their GAPs

Mishra

Lambright

2016

Biopolymers

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Summary Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bound inactive states under control of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which accelerate slow intrinsic rates of activation by nucleotide exchange and deactivation by GTP hydrolysis, respectively. Here we review developments leading to current understanding of intrinsic and GAP catalyzed GTP hydrolytic reactions in small GTPases from structural, molecular and chemical mechanistic perspectives. Despite the apparent simplicity of the GTPase cycle, the structural bases underlying the hallmark hydrolytic reaction and catalytic acceleration by GAPs are considerably more diverse than originally anticipated. Even the most fundamental aspects of the reaction mechanism have been challenging to decipher. Through a combination of experimental and in silico approaches, the outlines of a consensus view have begun to emerge for the best studied paradigms. Nevertheless, recent observations indicate that there is still much to be learned.

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Section: Small Gtpase Architecturementioning

confidence: 99%

Section: Chemical Kinetic Barriers and Reaction Pathwaysmentioning

confidence: 99%

Invited review: Small GTPases and their GAPs

Mishra

Lambright

2016

Biopolymers

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“…The small basis set M06/6-31G* was sufficient to reproduce the infrared experiments and the QM box only contained the nucleotide, Mg 2+ and its coordinating water molecules, enabling fast calculation of QM/MM spectra from a MM snapshot within one day on 8 parallel CPU cores. We calculated protonation of every phosphate group in GTP, including a protonation scheme that was suggested by neutron diffraction crystallography (24). The calculated NMR results of several protonations (1, 3, 1, 2) contradicted the experimental findings, indicating these protonation states were not present in the NMR experiments.…”

Section: Discussionmentioning

confidence: 88%

“…Protonation of -GTP caused deviation from the experimental NMR and IR spectra NMR and infrared spectra of 2-protonated GTP ( E-F. The protonation 2 corresponds to the protonation state that was proposed by the neutron diffraction crystal structure (24). The depicted structures that were obtained from QM/MM optimized snapshots of MD simulations (Fig.…”

Section: Atr-ftir Measurements Of Gtp Bound To Ras On a Model Membranementioning

confidence: 87%

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The protonation states of GTP and GppNHp in Ras proteins

Mann

Güldenhaupt

Nabers

et al. 2018

Journal of Biological Chemistry

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The small GTPase Ras transmits signals in a variety of cellular signaling pathways, most prominently in cell proliferation. GTP hydrolysis in the active center of Ras acts as a prototype for many GTPases and is the key to the understanding of several diseases, including cancer. Therefore, Ras has been the focus of intense research over the last decades. A recent neutron diffraction crystal structure of Ras indicated a protonated -guanylyl imidodiphosphate (-GppNHp) group, which has put the protonation state of GTP in question. A possible protonation of GTP was not considered in previously published mechanistic studies. To determine the detailed pre-hydrolysis state of Ras, we calculated infrared and NMR spectra from quantum mechanics/molecular mechanics (QM/MM) simulations and compared them with those from previous studies. Furthermore, we measured infrared spectra of GTP and several GTP analogs bound to lipidated Ras on a membrane system under near-native conditions. Our findings unify results from previous studies and indicate a structural model confirming the hypothesis that -GTP is fully deprotonated in the pre-hydrolysis state of Ras.

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Allosteric site variants affect GTP hydrolysis on Ras

Johnson,

Fetics,

Davis

et al. 2023

Protein Science

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RAS GTPases are proto‐oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP‐bound and GTP‐bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4 and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF‐RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP‐bound state of RAS.This article is protected by copyright. All rights reserved.

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Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate

Cited by 45 publications

References 58 publications

Invited review: Small GTPases and their GAPs

Invited review: Small GTPases and their GAPs

The protonation states of GTP and GppNHp in Ras proteins

Allosteric site variants affect GTP hydrolysis on Ras

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