Structural heterogeneity and substrate engagement mechanism of the bacterial proteasome activator Bpa

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. Bpa (bacterial proteasome activator) is essential for Mycobacterium tuberculosis virulence, yet its structural heterogeneity and mode of substrate engagement have remained poorly understood. Here, the authors combine hydrogen/deuterium exchange mass spectrometry and solution NMR spectroscopy to elucidate Bpa assembly and its interaction with a model substrate.