Discovering Targetable Conformation of RhoA Mutant by Integrating Native Mass Spectrometry, Ultraviolet Photodissociation, and X-ray Diffraction

作者：Haozhe Wu, Zheyi Liu, Haibo Jiang, Heng Zhao, Chen Dong, Yu Lu, Shijia Zu, Yongjie Guo, Can Lai, Pan Luo, Ke Xu, Yuxin Yang, Yafang Yang, Zhongya Sun, Qingcheng Huang, Huan Xiong, Lanxin Zhou, Yipan Luo, Yuhan Zeng, Daohai Du, Zhongjie Liang, Weilie Xiao, Shan Zhao, Weiqing Zhang, Yong Tang, Chunlei Xiao, Kaixian Chen, Xueming Yang, Fangjun Wang, Cheng Luo · 发表于：Journal of the American Chemical Society · 年份：2026 · DOI：10.1021/jacs.5c20067 · 被引用次数：2 · 研究领域：Protein Kinase Regulation and GTPase Signaling、Enzyme Structure and Function、Advanced Proteomics Techniques and Applications

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 2+ -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.