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Updated: Jul 18, 2025

Comparing the Affinity of GTPase-binding Proteins using Competition Assays
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Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Published on: October 8, 2015

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Allosteric site variants affect GTP hydrolysis on Ras

Christian W Johnson1, Susan K Fetics2, Kathleen P Davis2

  • 1Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA.

Protein Science : a Publication of the Protein Society
|August 24, 2023
PubMed

Insights

Manipulating the allosteric site of HRAS GTPase impacts GTP hydrolysis. Allosteric site closure increases HRAS GTP hydrolysis, while RAF kinase binding inhibits it, potentially stabilizing the GTP-bound state in cancer.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Signaling

Background:

  • RAS GTPases are key regulators of cell growth and proliferation, cycling between GDP- and GTP-bound states.
  • GTP hydrolysis by HRAS is crucial for signal termination and is potentially regulated by an allosteric site.

Purpose of the Study:

  • To investigate the functional connection between HRAS intrinsic GTP hydrolysis and the allosteric site.
  • To determine how manipulating the allosteric site affects HRAS GTPase activity.

Main Methods:

  • Site-directed mutagenesis to introduce hydrophobic mutations in the HRAS allosteric site.
  • X-ray crystallography to determine structural changes in HRAS mutants.
  • Enzymatic assays to measure GTP hydrolysis rates in solution.

Main Results:

  • Closure of the allosteric site in HRAS mutants correlated with increased intrinsic GTP hydrolysis.
  • Binding to RAF-RBD inhibited GTP hydrolysis in these HRAS mutants.
  • Mutant behavior may mimic tumor-associated mutations stabilizing the GTP-bound RAS state.

Conclusions:

  • The allosteric site and intrinsic GTP hydrolysis of HRAS are functionally linked.
  • RAF kinase binding can counteract the effects of allosteric site mutations on GTP hydrolysis.
  • Understanding these mechanisms may offer insights into RAS-driven oncogenesis.
Keywords:
HRASKRASRAFallosteric siteallosteryhydrolysismutationsmall GTPase

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