The ubiquitous CLC membrane transporters are unique in their ability to exchange anions for cations. Despite extensive study, there is no mechanistic model that fully explains their 2:1 Cl‒/H+ stoichiometric exchange mechanism. Here, we provide such a model. Using differential hydrogen-deuterium exchange mass spectrometry, cryo-EM structure determination, and molecular dynamics simulations, we uncovered new conformational dynamics in CLC-ec1, a bacterial CLC homolog that has served as a paradigm for this family of transporters. Simulations based on a cryo-EM structure at pH 3 revealed critical steps in the transport mechanism, including release of Cl‒ ions to the extracellular side, opening of the inner gate, and novel water wires that facilitate H+ transport. Surprisingly, these water wires occurred independently of Cl‒ binding, prompting us to reassess the relationship between Cl‒ binding and Cl‒/H+ coupling. Using isothermal titration calorimetry and quantitative flux assays on mutants with reduced Cl‒ binding affinity, we conclude that, while Cl‒ binding is necessary for coupling, even weak binding can support Cl‒/H+ coupling. By integrating our findings with existing literature, we establish a complete and efficient CLC 2:1 Cl‒/H+ exchange mechanism.

• Publication type
• Journal Article MeSH
• Preprint MeSH

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

• MeSH
• Sodium-Calcium Exchanger * chemistry   MeSH
• Mammals * MeSH
• Protein Structure, Secondary MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sodium-Calcium Exchanger * MeSH

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

• Keywords
• Antibody, Biosimilars, Hydrogen/deuterium exchange, Mass spectrometry, Protein structure and dynamics, Proteolysis,
• MeSH
• Deuterium MeSH
• Mass Spectrometry MeSH
• Antibodies * MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Hydrogen MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Antibodies * MeSH
• Hydrogen MeSH

African Trypanosomes have developed elaborate mechanisms to escape the adaptive immune response, but little is known about complement evasion particularly at the early stage of infection. Here we show that ISG65 of the human-infective parasite Trypanosoma brucei gambiense is a receptor for human complement factor C3 and its activation fragments and that it takes over a role in selective inhibition of the alternative pathway C5 convertase and thus abrogation of the terminal pathway. No deposition of C4b, as part of the classical and lectin pathway convertases, was detected on trypanosomes. We present the cryo-electron microscopy (EM) structures of native C3 and C3b in complex with ISG65 which reveal a set of modes of complement interaction. Based on these findings, we propose a model for receptor-ligand interactions as they occur at the plasma membrane of blood-stage trypanosomes and may facilitate innate immune escape of the parasite.

• MeSH
• Complement Activation MeSH
• Complement Pathway, Alternative MeSH
• Complement C3-C5 Convertases metabolism   MeSH
• Cryoelectron Microscopy MeSH
• Complement C3 * metabolism   MeSH
• Complement C5 metabolism   MeSH
• Humans MeSH
• Trypanosoma brucei gambiense * metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• C3 protein, human MeSH Browser
• Complement C3-C5 Convertases MeSH
• Complement C3 * MeSH
• Complement C5 MeSH

Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.

• Keywords
• Globin-coupled histidine kinase, Heme-containing oxygen sensors, Hydrogen/deuterium exchange, Ligand binding, Mass spectrometry, Protein conformational dynamics, Signal transduction,
• MeSH
• Deuterium MeSH
• Heme chemistry   MeSH
• Hemeproteins * MeSH
• Mass Spectrometry methods   MeSH
• Oxygen metabolism   MeSH
• Artificial Intelligence MeSH
• Deuterium Exchange Measurement methods   MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Heme MeSH
• Hemeproteins * MeSH
• Oxygen MeSH
• Hydrogen MeSH

Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.

• Keywords
• Ancestral luciferase, Hydrogen/deuterium exchange, LoopGrafter, Mass spectrometry, Protein dynamics, Protein engineering,
• MeSH
• Deuterium chemistry   MeSH
• Mass Spectrometry methods   MeSH
• Protein Conformation MeSH
• Deuterium Exchange Measurement * methods   MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Hydrogen MeSH

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein-DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

• Keywords
• DNA, Hydrogen/deuterium exchange, Protein–DNA binding, Structural mass spectrometry, Transcription factor,
• MeSH
• Data Analysis MeSH
• Chromatography, Liquid MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Transcription Factors MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Hydrogen Deuterium Exchange-Mass Spectrometry * methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA MeSH
• Transcription Factors MeSH

Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurosporacrassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.

• Keywords
• hydrogen/deuterium exchange mass spectrometry, lignocellulose degradation, lytic polysaccharide monooxygenase, oxidative amino acid modification, peptide bond cleavage, reactive oxygen species,
• MeSH
• Cellulose chemistry   MeSH
• Fungal Proteins chemistry   MeSH
• Spectrometry, Mass, Electrospray Ionization MeSH
• Mass Spectrometry MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Hydrogen-Ion Concentration MeSH
• Protein Conformation MeSH
• Oxygen chemistry   MeSH
• Lignin chemistry   MeSH
• Copper chemistry   MeSH
• Neurospora crassa enzymology   MeSH
• Oxidative Stress MeSH
• Oxidoreductases chemistry   MeSH
• Mixed Function Oxygenases chemistry   MeSH
• Polysaccharides chemistry   MeSH
• Reactive Oxygen Species chemistry   MeSH
• Substrate Specificity MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cellulose MeSH
• Fungal Proteins MeSH
• Oxygen MeSH
• Lignin MeSH
• lignocellulose MeSH Browser
• Copper MeSH
• Oxidoreductases MeSH
• Mixed Function Oxygenases MeSH
• Polysaccharides MeSH
• Reactive Oxygen Species MeSH

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.

• MeSH
• HeLa Cells MeSH
• Histones chemistry   metabolism   MeSH
• Humans MeSH
• Peptides metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Peptide Hydrolases metabolism   MeSH
• Proteomics methods   MeSH
• Recombinant Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histones MeSH
• Peptides MeSH
• Peptide Hydrolases MeSH
• Recombinant Proteins MeSH

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.

• MeSH
• Diet, Gluten-Free * MeSH
• Celiac Disease enzymology   immunology   therapy   MeSH
• Drosophila metabolism   MeSH
• Enzyme Therapy * MeSH
• Gliadin metabolism   MeSH
• Glutens metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Humans MeSH
• Mice, Inbred NOD MeSH
• Mice MeSH
• Protein Glutamine gamma Glutamyltransferase 2 MeSH
• GTP-Binding Proteins metabolism   MeSH
• Proteolysis MeSH
• Transglutaminases metabolism   MeSH
• Inflammation immunology   metabolism   prevention & control   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gliadin MeSH
• Glutens MeSH
• Protein Glutamine gamma Glutamyltransferase 2 MeSH
• GTP-Binding Proteins MeSH
• Transglutaminases MeSH