Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.05.01.722288

Resolving hidden stoichiometries in Bacterial proteasome activator (Bpa)-substrate complexes by cryo-EM and charge detection mass spectrometry

Davis, B. T. V.·Uday, A. B. B.·Haris, A.·Keszei, A. F. A.·Ujma, J.·Bruton, D.·Richardson, K.·Mazhab-Jafari, M.·Giles, K.·Zeytuni, N.·Vahidi, S.
View original source ↗Export metadata ↓

Abstract

Bpa (Bacterial proteasome activator) is a regulatory particle within the Mycobacterium tuberculosis (Mtb) proteasome system that 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 Mtb 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 both apo and HspR-bound Bpa. CDMS revealed a previously unreported undecameric apo species and defines 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. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/722288v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@be4bb6org.highwire.dtl.DTLVardef@15ccf73org.highwire.dtl.DTLVardef@379a2eorg.highwire.dtl.DTLVardef@6b7b90_HPS_FORMAT_FIGEXP M_FIG C_FIG

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Davis, B. T. V., Uday, A. B. B., Haris, A., Keszei, A. F. A., Ujma, J., Bruton, D., Richardson, K., Mazhab-Jafari, M., Giles, K., Zeytuni, N., Vahidi, S.. 2026-05-05. Resolving hidden stoichiometries in Bacterial proteasome activator (Bpa)-substrate complexes by cryo-EM and charge detection mass spectrometry. https://doi.org/10.64898/2026.05.01.722288

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Deep reinforcement learning-driven discovery of a MsbA-targeted small-molecule antibiotic for the treatment of Acinetobacter baumannii infection

Antibiotics with new mechanisms are highly pursued to address the threat of infections caused by drug-resistant Gram-negative bacteria. Targeting MsbA, a key protein of the lipopolysaccharide biosynthesis pathway, represents a promising strategy to discover new classes of antibiotics. However, currently available MsbA-targeted molecules either lack sufficient potency or have unfavorable properties, necessitating expansion of chemical space. In this study, we chose the most promising cerastecin Cpd 4 as the template, and used two Artificial Intelligence (AI)-based tools, i.e. Link-INVENT and AutoMolDesigner for molecular design, performed chemical derivatization and antibacterial activity evaluation, which led to the discovery of Y-11 (MIC for A. baumannii: 0.5 g/mL). Encouragingly, Y-11 showed equivalent potency to Cpd4 for carbapenem-resistant A. baumannii, and less cytotoxicity and hemolysis as well as lower spontaneous resistance frequency. In vivo efficacy study demonstrated that Y-11 could effectively reduce bacterial loads in the mice infected by A. baumannii. The following mechanism study including molecular dynamics simulation, biochemical assay, and transmission electron microscope (TEM) analysis suggested that Y-11 inhibited the transport of lipooligosaccharide and impaired the formation of outer membrane, probably by competitively binding to the substrate binding site of MsbA and modulating ATPase activity. Taken together, we have discovered a MsbA-targeted small molecule Y-11 via AI-driven drug design, which provides a foundation for future antibiotic development.

biochemistry↗
bioRxiv2026

Dynamic architecture of the Rixosome reveals mechanism of activation and ITS2 processing

Eukaryotic ribosome assembly requires the coordinated processing and extensive remodeling of pre-rRNAs. During late nuclear maturation of the 60S subunit, sequential removal of the internal transcribed spacer 2 (ITS2) is initiated by endonucleolytic cleavage at site C2 by the conserved Las1 nuclease. Las1 acts together with the kinase Grc3 and the Rix1 complex to form the Rixosome, which also functions in transcriptional regulation. However, the assembly of the Rixosome, its recruitment to pre-ribosomes, and its activation for ITS2 cleavage remain unclear. Here, we present cryo-EM structures of the human LAS1 complex, two structures of the isolated Rixosome and nine transition states of Rix1-bound pre-60S particles from Schizosaccharomyces pombe. These structures reveal a dynamic Rixosome architecture in which the heterotetrameric Las1 complex engages one or two copies of the Rix1 complex. Rix1 binding is highly flexible in the human Rixosome but rigid in the yeast complex. The isolated yeast Rixosome remains inactive, but binding to the pre-60S particle triggers a structural rearrangement that allows for substrate engagement and activation of the nuclease. Together, our results define the dynamic architecture of the Rixosome and provide a structural framework for ITS2 processing during nuclear maturation of the eukaryotic 60S ribosomal subunit.

biochemistry↗
bioRxiv2026

SGFP-Grid Split GFP Graphene Grids

Affinity graphene grids provide a promising approach for selective protein capture in cryo-EM. Here, we introduce a split-GFP graphene grid platform(SGFP-G), in which graphene-conjugated GFP 1-10 selectively captures GFP11 tagged proteins from low concentration samples or cell lysates. This platform enables rapid assessment of target protein enrichment and particle distribution before vitrification via fluorescence imaging, while the grid design positions captured proteins away from the graphene surface and air-water interface. We also introduce a unique strategy to minimize nonspecific protein adsorption, thereby improving the selective enrichment of target proteins on this grid. Using GFP11-tagged apoferritin, we demonstrate fluorescence guided protein capture and obtain a 2.58 [A] cryoEM reconstruction, establishing SGFP-G as an affinity grid platform for high resolution structural studies with reduced sample requirements.

biochemistry↗