Mechanisms of isoform-specific residue influence on GTP-bound HRas, KRas, and NRas

Volmar, Alicia Yvette; Guterres, Hugo; Zhou, Hao; Reid, Derion; Pavlopoulos, Spiro; Makowski, Lee; Mattos, Carla

doi:10.1016/j.bpj.2022.07.005

Cited by 6 publications

(5 citation statements)

References 55 publications

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“…A role of I93 in the global Ras dynamics has been suggested before from 15 N NMR experiments 24 ; 25, consistent with our structural observations of changes in conformations of this side chain in response to mutation of R97. Likewise, Y137 is more dynamic than suggested by the crystal structures alone, as it is a site of phosphorylation in HRAS by ABL kinase 26.…”

Section: Allosteric Site Mutants Stabilize the R-state Through Compen...supporting

confidence: 92%

Allosteric site variants affect GTP hydrolysis on RAS

Johnson

Fetics

Davis

et al. 2023

Preprint

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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 poorly understood mutations that occur in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.

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Section: Allosteric Site Mutants Stabilize the R-state Through Compen...supporting

confidence: 92%

Allosteric site variants affect GTP hydrolysis on RAS

Johnson

Fetics

Davis

et al. 2023

Preprint

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“…These two isoforms are well conserved between HRAS (95% sequence similarity), yet some amino acid substitutions exist near the allosteric site and network (Johnson et al, 2017 ). These isoforms differ subtly from HRAS in their dynamics and ability to promote GTP hydrolysis (Harrison et al, 2016 ; Johnson et al, 2017 ; Killoran & Smith, 2019 ; Parker et al, 2018 ; Volmar et al, 2022 ). Whether the allosteric site plays a role in these functional differences, and how the allosteric site interacts with oncogenic mutations in the active site, are necessary next steps to better understand these critically important enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…The active site of HRAS undergoes several conformational transitions once it becomes bound to GTP (Lu et al, 2016 ; Volmar et al, 2022 ). Here, we differentiate the sub‐states of state 2 relevant to our model of hydrolysis as the inactive “T‐state” (gray, Figure 1a ) and the catalytically active “R‐state” (green, Figure 1a ; Johnson & Mattos, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

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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“…Their differences lead to their differential membrane anchorage preferences, and altered interactions with varied stabilities with the same effectors. [198][199][200][201] Analysis of their dimer preferences demonstrates distinct behavior as well. 202 These point to differential distributions (propensities) of their conformational ensembles, which correlate with their altered preferred functions in different cell types.…”

Section: Splicing and Different Gene Isoforms May Share Ensembles Alt...mentioning

confidence: 99%