Related Experiment Video

Updated: Sep 9, 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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Time-Resolved Crystallography Reveals the Mechanisms of GTP hydrolysis for N-RAS and the Oncogenic Mutants G12C, G12V

Guowu Lin1, Paola Zinser-Peniche1, Xiaohong Zhou1

  • 1Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Biorxiv : the Preprint Server for Biology
|September 2, 2025
PubMed

Insights

Researchers visualized GTP hydrolysis in RAS proteins, revealing distinct reaction mechanisms for cancer-linked mutants. This structural insight into GTPase activity could guide new cancer drug discovery targeting specific intermediate states.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • RAS GTPases are crucial regulators of cell signaling, acting as molecular switches.
  • Mutations in RAS are prevalent in human cancers, often impairing GTP hydrolysis and leading to uncontrolled cell growth.
  • The precise mechanism of GTP hydrolysis, essential for RAS inactivation, has remained structurally elusive.

Purpose of the Study:

  • To structurally resolve the real-time mechanism of GTP hydrolysis in RAS proteins.
  • To investigate differences in GTP hydrolysis mechanisms between wild-type RAS and key oncogenic mutants.
  • To identify potential drug targets based on transient binding pockets during the hydrolysis reaction.

Main Methods:

  • Time-resolved crystallography using a photolabile caged-GTP substrate.
  • Capture and analysis of multiple reaction intermediates during GTP hydrolysis.
  • Structural comparison of N-RAS wild-type, oncogenic mutants (G12C, G12V, Q61L), and a fast hydrolytic mutant (Y32R).

Main Results:

  • Fifty-seven distinct reaction intermediates were captured, detailing the GTP hydrolysis pathway.
  • Common mechanistic elements were observed, including an initial open state, a closed Michaelis complex, and release of products.
  • Significant differences in reaction mechanisms and rates were identified between wild-type and mutant RAS proteins.
  • Cryptic binding pockets, state-dependent and unique to mutants, were discovered during hydrolysis.

Conclusions:

  • The study provides the first structural elucidation of the GTP hydrolysis mechanism in RAS proteins.
  • Understanding these mechanisms offers insights into the role of RAS mutations in cancer.
  • The identified state-dependent binding pockets represent promising targets for structure-based drug discovery against RAS-driven cancers.

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