Resolving Hidden Stoichiometriesin Bacterial ProteasomeActivator (Bpa)-Substrate Complexes by Cryo-EM and Charge DetectionMass Spectrometry

Bacterial proteasome activator (Bpa) is a regulatory particle within the Mycobacterium tuberculosis (Mtb) proteasome system that facilitates ATP-independent substrate engagement and delivery to the 20S core particle (CP) for degradation. The best characterized Bpa substrate is HspR, a transcriptional repressor of Mt stress-response genes whose Bpa-dependent degradation is required for pathogen virulence. However, the stoichiometry of the Bpa:HspR complex, the molecular mechanism of substrate engagement, and the heterogeneity of the resulting assemblies remain unclear. Here, we combine charge detection mass spectrometry (CDMS) and single-particle electron cryomicroscopy (cryo-EM) as complementary approaches to characterize apo and HspR-bound Bpa. CDMS revealed a previously unreported undecameric apo species and defined a Bpa12:HspR2 complex stoichiometry with minimal heterogeneity. Cryo-EM, performed without the employment of cross-linking reagents, independently confirmed the presence of undecameric Bpa in solution and localized substrate-associated density to the C-terminal H4 helix of Bpa. Together, these complementary single-particle approaches inform future efforts to target the Mtb proteasome system and provide new molecular insight into proteasomal substrate recognition in prokaryotes.