Abstract
Bacterial proteasomal activator (Bpa) is a regulatory particle of the Mycobacterium tuberculosis proteasome that facilitates the recruitment of substrates and their subsequent degradation by the 20S core particle. Substrate-bound structures of Bpa are unavailable, leaving its recruitment mechanism incompletely understood. Here, we use mass spectrometry and NMR to show that Bpa reversibly assembles into dodecamers from dimers/tetramers in a temperature-dependent manner in vitro, and map the oligomerization interfaces during assembly. To overcome the limitations posed by the poor solubility of natural Bpa substrates, we establish the DNA-binding domain of hTRF1 as a model substrate. We quantify the affinity and stoichiometry of the Bpa-hTRF1 interaction using methyl-TROSY NMR, identifying a 12 Bpa subunit: 3 hTRF1 binding ratio with micromolar affinity that is modulated by salt concentration. Our work maps the Bpa-hTRF1 interface at atomic resolution, identifies determinants of substrate engagement, and introduces a tractable substrate for dissecting proteasomal recognition in mycobacteria.
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Data availability
Mass spectrometry data are available from the MassIVE database as entry MSV000099239 (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=1e313073db3545efaf0ba91f95fa9a72) (HDX raw data). Source data are provided with this paper.
Code availability
The code used for SEC, HDX, and NMR analysis in this study has been deposited in the Zenodo database under accession code [https://zenodo.org/records/18498816] (Code deposition).
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Acknowledgements
B.T.V.D. acknowledges support from a Graduate Tuition Scholarship from the University of Guelph. Financial support was provided by Canadian Institutes of Health Research Project Grant PJT451412 to S.V. and a Foundation Grant FND-503573 to L.E.K., and by Discovery Grants from the Natural Sciences and Engineering Council of Canada RGPIN-2021-02843 to S.V and 024-03872 to L.E.K. MS data were recorded at the Mass Spectrometry Facility of the Advanced Analysis Centre, University of Guelph. We thank Dr. Dyanne Brewer (University of Guelph) for assistance with MS measurements. We thank Prof. Ashok Sekhar (Indian Institute of Science, Bangalore) for providing backbone resonance assignments of TRF1. We thank Dr. Taylor Forrester (University of Guelph) for help with the generation of the protein topology diagrams. We thank Dr. Algirdas Velyvis (University of Guelph) for guidance and helpful discussions.
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B.T.V.D., E.R., L.E.K., and S.V. initiated the project; B.T.V.D., E.R., L.E.K., and S.V. designed research; B.T.V.D., E.R., A.H., J.U., L.E.K., and S.V. performed research; B.T.V.D., E.R., A.H., J.U., D.B., K.R., K.G., L.E.K., and S.V. contributed new reagents/analytic tools; B.T.V.D., E.R., A.H., and S.V. analyzed data; B.T.V.D., E.R., L.E.K., and S.V. wrote the paper.
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A.H, J.U, D.B, K.R, and K.G. are employees of Waters Corporation, whose instrumentation was used in this study. Waters provided technical support, including access to software and, in the case of CD-MS, the prototype instrument itself, enabling nonconventional experiments to be performed. However, the company did not participate in experimental design, data interpretation, or manuscript preparation. The remaining authors declare no competing interests.
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Davis, B.T.V., Rennella, E., Haris, A. et al. Structural heterogeneity and substrate engagement mechanism of the bacterial proteasome activator Bpa. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69978-w
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DOI: https://doi.org/10.1038/s41467-026-69978-w
