Discovering Targetable Conformation of RhoA Mutant by Integrating Native Mass Spectrometry, Ultraviolet Photodissociation, and X-ray Diffraction
Pathogenic mutations in "undruggable" Ras superfamily proteins challenge drug development by inducing subtle, dynamic conformational changes. Here, we integrated X-ray crystallography with native mass spectrometry and ultraviolet photodissociation (nMS-UVPD) to reveal a cryptic conformation in the oncogenic Y42C mutant of RhoA. While crystallography alone resolved two ambiguous structures, nMS-UVPD determined the dominant conformation by directly mapping the mutant's conformational dynamics, identifying an enhanced Mg<sup>2+</sup>-locked conformation. We explored the mechanism of mutation impairing GTP hydrolysis. This state unmasks a previously hidden, druggable pocket adjacent to Cys42, guiding our identification of a covalent inhibitor. Our integrated approach establishes a roadmap for targeting pathogenic protein mutants previously considered "undruggable" due to their highly dynamic nature.
Authors
- Shan Zhao (ORCID: https://orcid.org/0000-0003-1829-7538)
- Xin Yang (ORCID: https://orcid.org/0000-0001-5936-486X)
- Huan Xiong (ORCID: https://orcid.org/0000-0002-7191-3259)
- Zheyi Liu (ORCID: https://orcid.org/0000-0003-0608-5482)
- Yipan Luo
- Haibo Jiang (ORCID: https://orcid.org/0000-0002-3515-4398)
- Zhongjie Liang (ORCID: https://orcid.org/0000-0003-0763-5407)
- Pingping Luo (ORCID: https://orcid.org/0000-0002-8277-2930)
- Can Lai
- Daohai Du
- Weiqing Zhang (ORCID: https://orcid.org/0000-0001-6518-4152)
- Ke Xu (ORCID: https://orcid.org/0000-0002-6602-3382)
- Zhongya Sun
- Z. H. Lu (ORCID: https://orcid.org/0000-0003-1350-1130)
- Yuhan Zeng
- Chen Dong (ORCID: https://orcid.org/0000-0001-7226-8055)
- Kaixian Chen
- Y. P. Guo (ORCID: https://orcid.org/0000-0002-8492-4969)
- Weilie Xiao
- Haozhe Wu
- Qingcheng Huang
- Lanxin Zhou
- Yu Lu
- Fangjun Wang
- Shijia Zu
- Chunlei Xiao
- Yafang Yang
- Yong Tang
- Heng Zhao
- Yuxin Yang
Institutions
- Guiyang Medical University (CN)
- Dalian Institute of Chemical Physics (CN)
- Zunyi Medical University (CN)
- Yunnan University (CN)
- Chinese Academy of Sciences (CN)
- Fudan University (CN)
- ShanghaiTech University (CN)
- Shanghai Institute of Materia Medica (CN)
- Drug Discovery Laboratory (Norway) (NO)
- Institute for Advanced Study (DE)
- Center for Systems Biology (US)
- Ensemble Therapeutics (United States) (US)
- University of Chinese Academy of Sciences (CN)
- Institute of Advanced Light Source Facilities Shenzhen (CN)
- State Key Laboratory of Drug Research
- Southern Medical University (CN)
Publication Details
- Journal
- Journal of the American Chemical Society
- Published
- 2026-03-16
- DOI
- https://doi.org/10.1021/jacs.5c20067
- Primary Topic
- Protein Kinase Regulation and GTPase Signaling
- Type
- article
- Field-Weighted Citation Impact
- 0.00