Resolving symmetry-masked allosteric cooperativity in the M. tuberculosis proteasome core particle

Authors

Turner, M., Wittlinger, V., Lento, C., Haris, A., Ujma, J., Bruton, D., Richardson, K., Giles, K., Bottcher, T., Wilson, D., Vahidi, S.

Abstract

The 20S proteasome core particle (CP) is a stacked 7-{beta}7-{beta}7-7 assembly in which the central {beta}-rings host fourteen catalytic active sites responsible for regulated protein degradation. Allosteric coupling between catalytic {beta}-subunits has been characterized in eukaryotic proteasomes, whose heteromeric {beta}-rings permit subunit-specific perturbation. In bacterial proteasomes, however, the {beta}-rings are homomeric, and any allosteric relationships between subunits with identical sequences have remained refractory to conventional ensemble-averaging structural methods, including to hydrogen/deuterium exchange mass spectrometry (HDX-MS). Here we show that orthosteric inhibitors paradoxically activate the Mycobacterium tuberculosis 20S CP at substoichiometric concentrations, revealing positive cooperativity between its {beta}-subunits. To dissect this cooperativity within the {beta}-ring, we co-assemble wild-type and catalytically inactive (T1A) {beta}-subunits into hybrid 20S CPs. We develop a probabilistic model relating bulk mixing ratios to the ensemble of hybrid 20S CP stoichiometries. Differential 15N-labelling of the wild-type subunits then resolves WT and T1A peptide signals by mass during HDX-MS, enabling subunit-resolved measurements within a single complex. Using this approach, we demonstrate that ligand binding at one {beta}-subunit remodels the conformational dynamics of binding-incompetent neighbours. Measuring deuterium uptake against ring composition identifies two allosteric routes: a lateral pathway from switch helix II to the active site of the adjacent intra-ring subunit, and an axial pathway connecting a loop at the {beta}-ring interface to the S pockets of the opposing ring. More broadly, this work establishes a framework for resolving symmetry-masked allostery in multi-subunit assemblies.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 04 Jul 2026.

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