Mass Spectrometry-Based Protein Footprinting Defines the Binding Pocket of Crotonylated H3K14 in the PHD1 Domain of BAF45D within the BAF Chromatin Remodeling Complex.

The BRG-/BRM-associated factor (BAF) chromatin remodeling complex is a central actor in transcription. One mechanism by which BAF affects gene expression is via its various histone mark readers, including double plant homeodomains (DPF), located in the BAF45D subunit. DPF domains recognize lysine acetyl and acylations, including crotonylation, localized at promoters and enhancers. Despite a significant degree of conservation between DPF domains, attempts to crystallize BAF45D with a crotonylated histone 3 peptide (H3K14Cr) were unsuccessful. In addition, recent cryoEM and modeled structures failed to define the Req domain of BAF45D, which is responsible for reading lysine modifications. Thus, the precise mechanism of crotonyl group recognition and binding by BAF45D within the BAF complex remains unclear. We turned to protein footprinting mass spectrometry to map the binding interface between H3K14Cr and BAF45D. This technique is able to demarcate protein-binding interfaces by modifying surface-accessible residues and is not limited by protein size or composition. Experiments performed in the isolated DPF domain of BAF45D (BAF45DDPF)-delineated H3K14Cr peptide binding across the PHD1 and PHD2 pockets. We observed markedly similar effects on the BAF45D subunit when assessing H3K14Cr binding in the purified full BAF complex. The ATPase motor, BRM, also displayed H3K14Cr-protected peptides in two separate domains that were subsequently evaluated in direct binding assays. These data confirm the BAF45D-crotonylamide interaction within its obligate complex and are the first to demonstrate H3K14Cr direct binding to BRM.

Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids.

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes.

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline.

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique.

Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting.

Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.

Small molecule inhibitors of 15-PGDH exploit a physiologic induced-fit closing system.

15-prostaglandin dehydrogenase (15-PGDH) is a negative regulator of tissue stem cells that acts via enzymatic activity of oxidizing and degrading PGE2, and related eicosanoids, that support stem cells during tissue repair. Indeed, inhibiting 15-PGDH markedly accelerates tissue repair in multiple organs. Here we have used cryo-electron microscopy to solve the solution structure of native 15-PGDH and of 15-PGDH individually complexed with two distinct chemical inhibitors. These structures identify key 15-PGDH residues that mediate binding to both classes of inhibitors. Moreover, we identify a dynamic 15-PGDH lid domain that closes around the inhibitors, and that is likely fundamental to the physiologic 15-PGDH enzymatic mechanism. We furthermore identify two key residues, F185 and Y217, that act as hinges to regulate lid closing, and which both inhibitors exploit to capture the lid in the closed conformation, thus explaining their sub-nanomolar binding affinities. These findings provide the basis for further development of 15-PGDH targeted drugs as therapeutics for regenerative medicine.

Multiplex Chemical Labeling of Amino Acids for Protein Footprinting Structure Assessment.

Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a "one-pot" reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois' reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as "one-pot" reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.

GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium.

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air-liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbß expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, ß, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbß gene expression were associated with lower FEV1 in asthma. Both Hbß knockdown and overexpression affected cell morphology. Hbß and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbß gene expression were associated with airflow obstruction. Hbß and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

Structurally distinct external solvent-exposed domains drive replication of major human prions.

There is a limited understanding of structural attributes that encode the iatrogenic transmissibility and various phenotypes of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Here we report the detailed structural differences between major sCJD MM1, MM2, and VV2 prions determined with two complementary synchrotron hydroxyl radical footprinting techniques-mass spectrometry (MS) and conformation dependent immunoassay (CDI) with a panel of Europium-labeled antibodies. Both approaches clearly demonstrate that the phenotypically distant prions differ in a major way with regard to their structural organization, and synchrotron-generated hydroxyl radicals progressively inhibit their seeding potency in a strain and structure-specific manner. Moreover, the seeding rate of sCJD prions is primarily determined by strain-specific structural organization of solvent-exposed external domains of human prion particles that control the seeding activity. Structural characteristics of human prion strains suggest that subtle changes in the organization of surface domains play a critical role as a determinant of human prion infectivity, propagation rate, and targeting of specific brain structures.

Integrative structural modeling of a multidomain polo-like kinase.

Polo-like kinase 1 (PLK1) is a key regulator and coordinator for mitotic signaling that contains two major functional units of a kinase domain (KD) and a polo-box domain (PBD). While individual domain structures of the KD and the PBD are known, how they interact and assemble into a functional complex remains an open question. The structural model from the KD-PBD-Map205PBM heterotrimeric crystal structure of zebrafish PLK1 represents a major step in understanding the KD and the PBD interactions. However, how these two domains interact when connected by a linker in the full length PLK1 needs further investigation. By integrating different sources of structural data from small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational sampling, here we report an overall architecture for PLK1 multidomain assembly between the KD and the PBD. Our model revealed that the KD uses its C-lobe to interact with the PBD via the site near the phosphopeptide binding site in its auto-inhibitory state in solution. Disruption of this auto-inhibition via site-directed mutagenesis at the KD-PBD interface increases its kinase activity, supporting the functional role of KD-PBD interactions predicted for regulating the PLK1 kinase function. Our results indicate that the full length human PLK1 takes dynamic structures with a variety of domain-domain interfaces in solution.

Voltage-gated potassium channel proteins and stereoselective S-nitroso-l-cysteine signaling.

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase-independent (sGC-independent) effects are stereoselective - that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) - and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvß2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.

Fast Protein Footprinting by X-ray Mediated Radical Trifluoromethylation.

Synchrotron radiolysis generates hydroxyl radicals (•OH) that are successful footprinting reagents. Here, we describe a new reagent for the synchrotron platform, the trifluoromethyl radical (•CF3). The radical is produced by •OH displacement of •CF3 from sodium triflinate (Langlois reagent). Upon X-ray beam exposure, the reagent labels proteins extensively without any additional chemicals on a millisecond or shorter time scale. The •CF3 is comparably reactive to •OH and produces footprinting information that complements that of •OH alone. This reagent in combination with •OH should enable novel chemistry for protein footprinting on the synchrotron platform.

Selective PP2A Enhancement through Biased Heterotrimer Stabilization.

Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, "footprinting" studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver.

Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells.

BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length.

Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein-protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.

A Metastable Contact and Structural Disorder in the Estrogen Receptor Transactivation Domain.

The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins, mediates the receptor's transactivation function. However, an accurate molecular dissection of NTD's structure-function relationships remains elusive. Here, we show that the NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion on chemical denaturation. By combining small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map revealing metastable long-range contacts, e.g., between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) nuclear magnetic resonance that mutations near I33 alter 19F chemical shifts at S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of how specific contact metastability mediates critical functions of disordered proteins.

Structural attributes of mammalian prion infectivity: Insights from studies with synthetic prions.

Prion diseases are neurodegenerative disorders that affect many mammalian species. Mammalian prion proteins (PrPs) can misfold into many different aggregates. However, only a small subpopulation of these structures is infectious. One of the major unresolved questions in prion research is identifying which specific structural features of these misfolded protein aggregates are important for prion infectivity in vivo Previously, two types of proteinase K-resistant, self-propagating aggregates were generated from the recombinant mouse prion protein in the presence of identical cofactors. Although these two aggregates appear biochemically very similar, they have dramatically different biological properties, with one of them being highly infectious and the other one lacking any infectivity. Here, we used several MS-based structural methods, including hydrogen-deuterium exchange and hydroxyl radical footprinting, to gain insight into the nature of structural differences between these two PrP aggregate types. Our experiments revealed a number of specific differences in the structure of infectious and noninfectious aggregates, both at the level of the polypeptide backbone and quaternary packing arrangement. In particular, we observed that a high degree of order and stability of ß-sheet structure within the entire region between residues ∼89 and 227 is a primary attribute of infectious PrP aggregates examined in this study. By contrast, noninfectious PrP aggregates are characterized by markedly less ordered structure up to residue ∼167. The structural constraints reported here should facilitate development of experimentally based high-resolution structural models of infectiosus mammalian prions.

Multidomain architecture of estrogen receptor reveals interfacial cross-talk between its DNA-binding and ligand-binding domains.

Human estrogen receptor alpha (hERα) is a hormone-responsive nuclear receptor (NR) involved in cell growth and survival that contains both a DNA-binding domain (DBD) and a ligand-binding domain (LBD). Functionally relevant inter-domain interactions between the DBD and LBD have been observed in several other NRs, but for hERα, the detailed structural architecture of the complex is unknown. By utilizing integrated complementary techniques of small-angle X-ray scattering, hydroxyl radical protein footprinting and computational modeling, here we report an asymmetric L-shaped "boot" structure of the multidomain hERα and identify the specific sites on each domain at the domain interface involved in DBD-LBD interactions. We demonstrate the functional role of the proposed DBD-LBD domain interface through site-specific mutagenesis altering the hERα interfacial structure and allosteric signaling. The L-shaped structure of hERα is a distinctive DBD-LBD organization of NR complexes and more importantly, reveals a signaling mechanism mediated by inter-domain crosstalk that regulates this receptor's allosteric function.

Structural insights into the molecular mechanism of mouse TRPA1 activation and inhibition.

Pain, though serving the beneficial function of provoking a response to dangerous situations, is an unpleasant sensory and emotional experience. Transient receptor potential ankyrin 1 (TRPA1) is a member of the transient receptor potential (TRP) cation channel family and is localized in "nociceptors," where it plays a key role in the transduction of chemical, inflammatory, and neuropathic pain. TRPA1 is a Ca2+-permeable, nonselective cation channel that is activated by a large variety of structurally unrelated electrophilic and nonelectrophilic chemical compounds. Electrophilic ligands are able to activate TRPA1 channels by interacting with critical cysteine residues on the N terminus of the channels via covalent modification and/or disulfide bonds. Activation by electrophilic compounds is dependent on their thiol-reactive moieties, accounting for the structural diversity of the group. On the other hand, nonelectrophilic ligands do not interact with critical cysteines on the channel, so the structural diversity of this group is unexplained. Although near-atomic-resolution structures of TRPA1 were resolved recently by cryo-electron microscopy, in the presence of both agonists and antagonists, detailed mechanisms of channel activation and inhibition by these modulators could not be determined. Here, we investigate the effect of both electrophilic and nonelectrophilic ligands on TRPA1 channel conformational rearrangements with limited proteolysis and mass spectrometry. Collectively, our results reveal that channel modulation results in conformational rearrangements in the N-terminal ankyrin repeats, the pre-S1 helix, the TRP-like domain, and the linker regions of the channel.