Discovering Targetable Conformation of RhoA Mutant by Integrating Native Mass Spectrometry, Ultraviolet...

Area of Science:

• Biochemistry and structural biology
• Molecular oncology
• Drug discovery

Background:

• Ras superfamily proteins, including RhoA, are frequently mutated in cancer.
• Pathogenic mutations often induce subtle, dynamic conformational changes, making them difficult targets for drug development.
• The Y42C mutation in RhoA is oncogenic but considered
• undruggable
• due to its complex conformational dynamics.

Purpose of the Study:

• To investigate the conformational dynamics of the oncogenic RhoA Y42C mutant.
• To identify novel druggable pockets in previously intractable protein mutants.
• To develop a strategy for targeting
• undruggable
• cancer-associated protein mutations.

Main Methods:

• Integrated X-ray crystallography with native mass spectrometry and ultraviolet photodissociation (nMS-UVPD).
• nMS-UVPD was used to directly map the conformational dynamics of the RhoA Y42C mutant.
• Computational analysis to understand the mechanism of impaired GTP hydrolysis.

Main Results:

• nMS-UVPD revealed a dominant, enhanced Mg²⁺-locked conformation of RhoA Y42C, which was ambiguous by crystallography alone.
• This conformation unmasks a previously hidden, druggable pocket adjacent to Cys42.
• A covalent inhibitor targeting this pocket was identified.

Conclusions:

• The integrated nMS-UVPD and crystallography approach successfully characterized the dynamics of a challenging protein mutant.
• A novel druggable pocket was identified in the oncogenic RhoA Y42C mutant.
• This study provides a roadmap for targeting previously
• undruggable
• pathogenic protein mutants in cancer therapy.