Biology subjects

Vahidi, S.

Publications and source records attributed to Vahidi, S..

14 recordsLinked to original sources

bioRxiv2026

Navigating the oligomeric landscape of the periplasmic stress response protease-chaperone DegP with charge detection mass spectrometry

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.

biochemistry↗

bioRxiv2026

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

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. Significance StatementThe 20S proteasome is essential for the survival and virulence of Mycobacterium tuberculosis, yet how its catalytic sites communicate has been difficult to study because the bacterial enzyme is built from identical subunits whose signals are indistinguishable by conventional methods. Here we find that blocking only a fraction of these sites by inhibitors paradoxically activates the enzyme, revealing positive cooperativity between neighbouring subunits. To trace the communication pathways, we developed an isotopic-coding strategy that enables hydrogen/deuterium exchange mass spectrometry report on individual subunits within a single symmetric complex. This approach maps how ligand binding at one subunit reshapes its neighbours and, more broadly, provides a general framework for dissecting allostery in homomeric molecular machines whose symmetry has long obscured it.

biochemistry↗

bioRxiv2026

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

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

biochemistry↗

bioRxiv2026

Structural basis of substrate recognition for proteasome degradation by prokaryotic ubiquitin-like protein ligase PafA

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.

biochemistry↗

bioRxiv2025

A universal buffer system for native LC-MS analysis of antibody-based therapeutics

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, pKa 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, pKa 8.9), 2,2-difluoroethylamine (DFEA, pKa 7.2), and 2,2,2-trifluoroethylamine (TFEA, pKa 5.5), 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 non-volatile 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 while maintaining compatibility with mass spectrometry.

biochemistry↗

bioRxiv2025

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.

biochemistry↗

bioRxiv2025

Characterization of conformational dynamics and allostery of the catalytic domain of human mitochondrial YME1L protease

Mitochondrial proteostasis is essential to maintain cellular function and survival. YME1L protease is an important contributor to proteostasis which belongs to the AAA+ (ATPases Associated with diverse cellular Activities) protease family and is anchored to the inner mitochondrial membrane. YME1L plays a pivotal role in mitochondrial protein quality control by selectively degrading misfolded and native proteins. The precise mechanisms by which nucleotide binding and hydrolysis influence YME1Ls conformational dynamics, proteolytic activity, and stability remain unclear. Here we characterize the conformational dynamics and allosteric regulation 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 Zn{superscript 2} binding stabilizes the protease domain. We also reveal novel long-range allostery between the AAA+ and protease domains of YME1L, mediated by a critical salt bridge on the inter-domain interface. We show the importance of the salt bridge 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 modification- and heat-induced aggregation. These findings explain the nucleotide-driven regulation of YME1L and provide novel insights into understanding its proteolytic activity and structural stability under physiological and stress conditions.

biophysics↗

bioRxiv2024

Probing the structural heterogeneity of Pup ligase PafA using H/D exchange mass spectrometry

The Pup-proteasome system (PPS) is a unique bacterial proteolytic pathway found in some bacterial species, including in Mycobacterium tuberculosis, that plays a vital role in maintaining proteome integrity and survival during infection. Pupylation is the process of tagging substrates with Pup for degradation and is catalyzed by PafA, the sole Pup ligase in bacteria. However, how PafA interacts with diverse targets and its oligomeric state remain poorly understood. Although X-ray crystal structures have characterized PafA as a domain-swapped dimer, it is widely regarded as functionally active in its monomeric form. It remains to be established whether PafA dimerizes in solution, and how dimerization influences its function. In this study, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) alongside complementary biophysical techniques to explore the oligomeric states and conformational dynamics of PafA. We show that recombinantly-produced PafA exists in a monomeric and a domain-swapped dimeric state in solution. While PafAdimer is stabilized by nucleotide, it predominately resides in a catalytically inactive conformation. Our HDX-MS highlighted regions throughout the N- and C-terminal domains that facilitate the PafA dimerization process. HDX-MS also revealed nucleotide binding induces global conformational changes on the PafAmonomer, underscoring the structural plasticity of this promiscuous enzyme. Our findings provide insight into the structure-function-dynamics relationship of PafA, shedding light on how its oligomeric states and conformational flexibility might influence its role in the PPS.

biochemistry↗

bioRxiv2024

Mechanism of allosteric activation in human mitochondrial ClpP protease

Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently-discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remain elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present the first structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where sub-stoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement. Significance statementHuman ClpP protease is essential for maintaining mitochondrial protein quality by degrading damaged proteins. In cancers like acute myeloid leukemia (AML), ClpP is overexpressed, and inhibiting it causes cancer cell death by disrupting mitochondrial function. Conversely, activating ClpP with small molecules, such as ONC201, also leads to cancer cell death by impairing mitochondrial respiration. However, the structural details of ClpP activation have been elusive. Our research presents the first structures of human ClpP in its active state, revealing a novel activation mechanism where inhibitors unexpectedly trigger activity through allosteric changes. These insights provide a foundation for designing targeted therapies for AML and other diseases where ClpP plays a crucial role.

biochemistry↗

bioRxiv2024

HDgraphiX: A web-based tool for visualization of hydrogen deuterium exchange mass spectrometry data

SummaryHydrogen deuterium exchange mass spectrometry (HDX-MS) investigates protein structural changes by measuring deuterium incorporation into the protein amide backbone. Due to richness of information provided on protein conformational dynamics, HDX-MS data can be challenging to visualize effectively. To address this, we have developed HDgraphiX, a web-based tool that visualizes HDX data by processing outputs from two popular analysis software packages, DynamX (Waters Corp.) and HDExaminer (Sierra Analytics Inc). HDgraphiX performs statistical analyses, filters data based on statistical significance and presents the results in several forms of user-friendly publication-quality heatmaps (Chiclet plots). Unique features of this tool include the generation of Woods plots, volcano plots, and PyMOL colouring scripts, which are used to map deuterium uptake differences onto protein structures. Additionally, HDgraphiX offers numerous advanced options for customizing data processing and plotting without the need for manual data editing. Availability and ImplementationHDgraphiX is available free of charge for all users at https://hdgraphix.net, the Python script and HTML template are deposited at https://github.com/KentV-UofG/HDgraphiX.

biochemistry↗

bioRxiv2024

Structural basis for allosteric regulation of the proteasome core particle

Intracellular protein degradation is vital across all domains of life1. In eukaryotes, the ubiquitin proteasome system performs most non-lysosomal protein degradation and influences numerous cellular processes. Some bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), encode a proteasome system that selectively degrades damaged or misfolded proteins crucial for the pathogens survival within host macrophages2-7. Consequently, the 20S core particle (CP), the central component of the proteasome system, has emerged as a viable target for tuberculosis treatment strategies2,8-10. Both eukaryotic and Mtb proteasome systems are allosterically regulated11-13, yet the specific conformations involved have not been captured in high-resolution structures to date. Here we present the first structure of Mtb 20S CP, and indeed any 20S CP, in an inactive state called 20SOFF, distinguished from the canonical active state, 20SON, by the conformation of switch helices I and II. The rearrangement of these helices collapses the S1 pocket, effectively inhibiting substrate binding. The switch helices are conserved and regulate the activity of HslV protease, the proteasomes ancestral enzyme in bacteria, and a diverse family of serine/threonine protein phosphatases. Our results highlight the potential of harnessing allostery to develop therapeutics against the 20S CP in Mtb and eukaryotic systems.

biochemistry↗

bioRxiv2022

Structure of mycobacterial respiratory Complex I

Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found at low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the "orphan" two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel and suggests that menaquinone interacts more extensively than ubiquinone with a key catalytic histidine residue in the enzyme.

biochemistry↗

bioRxiv2019

An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as novel antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of anti-TB therapeutics that target the Clp system. Here we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Cryo-EM structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key {beta}-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that upon binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function, and shows extensive allosteric interactions involving both intra- and inter-ring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis. Significance StatementThe MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis in Mycobacterium tuberculosis, the causative agent of TB. Antibiotics that selectively kill both dormant and growing drug-resistant populations of M. tuberculosis by disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health.

biophysics↗

bioRxiv2019

A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery

The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here we present cryo-EM structures of the substrate-bound ClpXP complex from Neisseria meningitidis at 2.3 to 3.3 [A] resolution. The structures allow development of a model in which the cyclical hydrolysis of ATP is coupled to concerted motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.

biophysics↗