Abstract
Protein phosphorylation is a pivotal post-translational modification modulating various cellular processes. In Gram-positive bacteria, the protein arginine kinase McsB, along with its activator McsA, has a key role in labeling misfolded and damaged proteins during stress. However, the activation mechanism of McsB by McsA remains elusive. Here we report the cryo-electron microscopy structure of a tetrameric McsA–McsB complex at 3.41 Å resolution. Biochemical analysis indicates that the homotetrameric assembly is essential for McsB’s kinase activity. The conserved C-terminal zinc finger of McsA interacts with an extended loop in McsB, optimally orienting a critical catalytic cysteine residue. In addition, McsA binding decreases the CtsR’s affinity for McsB, enhancing McsB’s kinase activity and accelerating the turnover rate of CtsR phosphorylation. Furthermore, McsA binding also increases McsB’s thermostability, ensuring its activity under heat stress. These findings elucidate the structural basis and activation mechanism of McsB in stress response.
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Data availability
Atomic coordinates and cryo-EM maps of McsA–McsB complex structure have been deposited in the PDB with accession code 8GQD (https://www.rcsb.org/structure/8GQD) and EMDB with code EMD-34200 (https://www.ebi.ac.uk/emdb/EMD-34200). All other source data are included in the paper as source or Supplementary Data 1 and 2. PDB codes 6FH1 (https://www.rcsb.org/structure/6FH1) and 3JPZ (https://www.rcsb.org/structure/3JPZ) are obtained from the PDB database. Source data are provided with this paper.
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Acknowledgements
We are grateful to C. Fu (Xiamen University) for providing the pArg antibody. Cryo-EM data were collected on Can Cong at SKLB West China Cryo-EM Center and processed at SKLB Duyu High-Performance Computing Center of West China Hospital. This work was supported by the National Natural Science Foundation of China (22077142 and 22022706 to W.X., 22293053 and 92353301 to Z.-W.M., and 32070049 and 32222040 to Z.S.), the National Key Research and Development Program of China (2022YFA1104900 to W.X., 2022YFB3804502 to Z.-W.M. and 2022YFC2303700 to Z.S.), the Fundamental Research Funds for the Central Universities (23xkjc020 to W.X.) and the 1.3.5 Project for Disciplines Excellence of West China Hospital (ZYYC21006 to Z.S.).
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K.L., B.L., X.T., P.Z., G.W., S.G., W.X., Z.S. and Z.-W.M. conceived the study and designed or supervised the experiments. M.A. and B.Z. performed the MD calculation and data analysis. X.X. performed the native mass studies. X.M. performed the AUC experiments. Y.L., H.L., Y.X. and Z.Z. helped with biochemical and structural analyses. J.N. and C.W. performed the mass photometry experiments. K.L., X.T., W.X. and Z.-W.M. drafted the manuscript, and all authors read and edited the manuscript and approved the final version.
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Lu, K., Luo, B., Tao, X. et al. Complex structure and activation mechanism of arginine kinase McsB by McsA. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01720-3
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DOI: https://doi.org/10.1038/s41589-024-01720-3
