Assembly and substrate engagement mechanism of the bacterial proteasome activator Boa

The bacterial proteasomal activator Bpa (Rv3780) is an ATP-independent regulatory particle of the Mycobacterium tuberculosis proteasome system. Bpa recruits substrates as a dodecamer and triggers the gate opening of the proteasome 20S core particle; however, the structural basis for its oligomerization and substrate recognition remains unclear. Here, we define the temperature-sensitive oligomerization mechanism of Bpa and elucidate its interaction with a non-native substrate. Using size-exclusion chromatography, charge detection mass spectrometry, and pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS), we show that Bpa reversibly assembles into a dodecameric ring from dimeric and tetrameric species in a temperature-dependent manner. We used HDX-MS to map the oligomerization interfaces during Bpa assembly. Methyl transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments and site-specific truncations further validate the existence of discrete tetrameric and dodecameric states. To overcome the limitations posed by the poor solubility of the native substrates of Bpa, we establish the DNA-binding domain of hTRF1 as a surrogate substrate. Bpa binds hTRF1 and mediates its degradation in a 20S CP-dependent manner. 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 NMR-based mapping experiments pinpoint the interaction surfaces on both Bpa and hTRF1, revealing key hydrophobic residues that mediate substrate engagement. This work uncovers a thermosensitive switch regulating Bpa oligomerization and activity and introduces a tractable substrate for dissecting proteasomal recognition in M. tuberculosis.