Siavash Vahidi

ClpP protease maintains proteostasis in human mitochondria and is overexpressed in cancers such as acute myeloid leukemia (AML). While activators such as dordaviprone (ONC201) have reached the clinic, the development of ClpP inhibitors has been hampered by paradoxical activation upon orthosteric inhibitor binding. Here, we report a series of quinazolinone-based compounds that allosterically inhibit human ClpP with nanomolar potency. Cryo-EM and H/D exchange mass spectrometry reveal that our lead compound binds sub-stoichiometrically to a distal hydrophobic pocket ~30 Å from the active sites, disrupts a conserved inter-subunit cluster, and induces a conformational change that misaligns the catalytic triad, thereby inactivating ClpP. The binding site of our quinazolinone-based inhibitors partially overlaps with that of the activator dordaviprone. In a xenograft mouse model of leukemia, our lead compound significantly reduced tumor burden following short-term oral administration. Our findings identify a regulatory hotspot in human ClpP and a mechanistically distinct inhibition strategy.

DegP is a periplasmic protease-chaperone essential for protein quality control and virulence factor trafficking in Gram-negative bacteria. In its apo form, DegP adopts a dynamic ensemble of oligomers derived from trimer building blocks through two competing self-assembly pathways. Upon engaging client proteins, apo DegP oligomers redistribute into discrete cage structures inside which the clients are encapsulated. The cage ensembles formed depend on the size of the bound clients, and notably can include 12mers, 24mers, and 60mers. Previous studies mapped the DegP oligmeric landscape using dynamic light scattering, analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and electron cryomicroscopy, modalities which in general report ensemble averages and often cannot directly delineate closely related coexisting species. Here, we apply charge detection mass spectrometry (CDMS) to directly measure the masses of individual DegP ions, resolving the complete oligomeric distribution in the absence and presence of four clients of increasing size. We reveal previously undetected odd-numbered oligomers and quantify the relative abundance of each assembly. Through heat-cool cycling CDMS experiments, we track cage distribution changes and reveal client protection and refolding, providing a direct view of the chaperone capabilities of DegP. These results further establish CDMS as a powerful single-molecule tool for dissecting heterogeneous protein assembly landscapes.

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.

The 20S proteasome core particle (CP) is a stacked α ₇ -β ₇ -β ₇ -α ₇ assembly in which the central β-rings host fourteen catalytic active sites responsible for regulated protein degradation. Allosteric coupling between catalytic β-subunits has been characterized in eukaryotic proteasomes, whose heteromeric β-rings permit subunit-specific perturbation. In bacterial proteasomes, however, the β-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 β-subunits. To dissect this cooperativity within the β-ring, we co-assemble wild-type and catalytically inactive (T1A) β-subunits into hybrid 20S CPs. We develop a probabilistic model relating bulk mixing ratios to the ensemble of hybrid 20S CP stoichiometries. Differential ¹⁵ N-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 β-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 β-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.

Bpa ( B acterial p roteasome a ctivator) 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 Bpa ₁₂ :HspR ₂ 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:

Selective protein degradation in some bacteria is performed by the Pup-proteasome system, in which the ligase PafA tags hundreds of substrates for proteasomal degradation. How a single enzyme achieves such broad substrate specificity in the absence of conserved sequence motifs has remained unclear. Here we determine structures of PafA in complex with a pupylated substrate and show that substrate recognition is mediated by a minimal and highly distributed interface. PafA samples an ensemble of closely related conformations that collectively position the target lysine residue for modification. This recognition mechanism arises from a combination of structured contacts and dynamic elements on both the enzyme and substrate, enabling geometric compatibility rather than sequence-specific interactions. These findings reveal an ensemble-driven mechanism of molecular recognition that explains how broad substrate specificity is achieved and provides a framework for understanding selective protein degradation in prokaryotes.

Liquid chromatography coupled to mass spectrometry (LC–MS) is a powerful analytical technique for analyzing biological macromolecules. A long-standing challenge has been applying LC–MS at physiological pH under native conditions using volatile buffers. The predominant “buffer” used, ammonium acetate (AmAc, pK_a 4.75 for acetic acid and 9.25 for ammonium), does not offer sufficient buffering capacity in the physiological pH range of 7.0–7.4. To address this, we evaluated a set of fluorinated ethylamines, 2-fluoroethylamine (MFEA), 2,2-difluoroethylamine (DFEA), and 2,2,2-trifluoroethylamine (TFEA), producing conjugate acids with pK_a values of 8.9, 7.2, and 5.5, respectively, that together provide buffering across the 4.5–9.8 pH range. We show that protein separations on strong cation- and anion-exchange resins in these volatile mobile phases perform comparably to traditional nonvolatile buffers, with similar elution profiles and analyte elution ranking, albeit with slightly broader peaks. Using fully volatile gradients of pH or ionic strength, we chromatographically resolved charge variants of protein analytes such as mAbs and bovine serum albumin. For many of the eluting LC peaks, we obtained high-resolution mass spectra capable of resolving glycoforms of antibodies. Hydrophobic interaction chromatography (HIC) in volatile mobile phases preserved native separation order and further resolved drug-to-antibody ratio (DAR) species of the antibody-drug conjugate brentuximab-vedotin. For each chromatography modality we further compare innovator and biosimilar antibodies, demonstrating the reproducibility of results in the proposed volatile compounds. Together, our results establish fluorinated ethylamines, in combination with ammonium acetate, as a universal volatile buffer system for native LC–MS, broadly applicable across major chromatographic modalities.

Most enzyme reactions occur in aqueous solution, but catalytic activity is often retained in nonaqueous solvents. It is an intriguing question whether enzymes can also remain active in the gas phase, without any solvent. Here, we address this question by examining electrosprayed [L·NAG₅ + 8H]⁸⁺ complexes (L = lysozyme, NAG_n = N-acetyl glucosamine oligomer, a lysozyme substrate in solution). Collisional activation triggered cleavage of NAG₅ into NAG₄ and a monomeric NAG unit. The reaction proceeded via a [L·NAG₄ + 8H]⁸⁺ enzyme·product complex, confirming that cleavage took place while NAG₅ was bound to lysozyme. We demonstrate that the reaction exhibits hallmarks of enzyme catalysis, including the participation of specific active site residues (E35 and D52), substrate specificity, and a lowered activation energy. The gas-phase products differ from those in solution, as dictated by the absence of water. Our experimental findings are bolstered by mobile-proton molecular dynamics simulations of gaseous lysozyme complexes with substrates and products. In conclusion, this work for the first time demonstrates that an enzyme can be catalytically active in vacuo. While it is yet to be determined if this finding can be generalized to other enzymes, our study may provide a stepping stone to novel enzyme-catalyzed (bio)molecular transformations in the absence of a solvent.

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.

Mitochondrial proteostasis is essential to maintain cellular function and survival. YME1L is a membrane-anchored AAA+ (ATPases Associated with diverse cellular Activities) family protease and plays a pivotal role in mitochondrial proteostasis by selectively degrading misfolded and native proteins. The precise mechanisms by which nucleotide binding and hydrolysis influence YME1L’s conformational dynamics, proteolytic activity, and stability remain unclear. Here, we characterize the conformational dynamics of the YME1L catalytic domain. Using a hexameric soluble YME1L construct, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) spectroscopy to demonstrate that nucleotide binding reduces the backbone flexibility and modulates the side-chain dynamics of the AAA+ domain, while Zn²⁺ binding stabilizes the protease domain. We also reveal long-range functional crosstalk between the AAA+ and protease domains of YME1L. We use functional assays to show the importance of a salt bridge between the AAA+ and protease domains in facilitating ATP-dependent substrate degradation by YME1L. Additionally, we show that ATP binding stabilizes the structure of the catalytic domain of YME1L and protects it from chemical- and heat-induced aggregation. These findings explain the nucleotide-driven regulation of YME1L and provide insights into our understanding of its proteolytic activity and structural stability under stress conditions.