Hydrogen-deuterium Exchange Mass Spectrometry Experiments Research Articles

The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

Deep Learning Enables Automatic Correction of Experimental HDX-MS Data with Applications in Protein Modeling.

Observed mass shifts associated with deuterium incorporation in hydrogen-deuterium exchange mass spectrometry (HDX-MS) frequently deviate from the initial signals due to back and forward exchange. In typical HDX-MS experiments, the impact of these disparities on data interpretation is generally low because relative and not absolute mass changes are investigated. However, for more advanced data processing including optimization, experimental error correction is imperative for accurate results. Here the potential for automatic HDX-MS data correction using models generated by deep neural networks is demonstrated. A multilayer perceptron (MLP) is used to learn a mapping between uncorrected HDX-MS data and data with mass shifts corrected for back and forward exchange. The model is rigorously tested at various levels including peptide level mass changes, residue level protection factors following optimization, and ability to correctly identify native protein folds using HDX-MS guided protein modeling. AI is shown to demonstrate considerable potential for amending HDX-MS data and improving fidelity across all levels. With access to big data, online tools may eventually be able to predict corrected mass shifts in HDX-MS profiles. This should improve throughput in workflows that require the reporting of real mass changes as well as allow retrospective correction of historic profiles to facilitate new discoveries with these data.

Structural flexibility and heterogeneity of recombinant human glial fibrillary acidic protein (GFAP).

Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3-dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX-MS experiments show an overall decrease in H-bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX-MS results with the predicted GFAP-dimer structure by AlphaFold-Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices.

Small Molecule Screen Identifies Non-catalytic USP3 Chemical Handle.

Zinc-finger ubiquitin-binding domains (ZnF-UBDs) are noncatalytic domains mostly found in deubiquitylases (DUBs) such as USP3. They represent an underexplored opportunity for the development of deubiquitylase-targeting chimeras (DUBTACs) to pharmacologically induce the deubiquitylation of target proteins. We previously showed that ZnF-UBDs are ligandable domains. Here, a focused small molecule library screen against a panel of 11 ZnF-UBDs led to the identification of compound 59, a ligand engaging the ZnF-UBD of USP3 with a KD of 14 μM. The compound binds the expected C-terminal ubiquitin binding pocket of USP3 as shown by hydrogen-deuterium exchange mass spectrometry experiments and does not inhibit the cleavage of K48-linked diubiquitin by USP3. As such, this molecule is a chemical starting point toward chemical tools that could be used to interrogate the function of the USP3 Znf-UBD and the consequences of recruiting USP3 to ubiquitylated proteins.

An HIV-1 broadly neutralizing antibody overcomes structural and dynamic variation through highly focused epitope targeting

The existence of broadly cross-reactive antibodies that can neutralize diverse HIV-1 isolates (bnAbs) has been appreciated for more than a decade. Many high-resolution structures of bnAbs, typically with one or two well-characterized HIV-1 Env glycoprotein trimers, have been reported. However, an understanding of how such antibodies grapple with variability in their antigenic targets across diverse viral isolates has remained elusive. To achieve such an understanding requires first characterizing the extent of structural and antigenic variation embodied in Env, and then identifying how a bnAb overcomes that variation at a structural level. Here, using hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and quantitative measurements of antibody binding kinetics, we show that variation in structural ordering in the V1/V2 apex of Env across a globally representative panel of HIV-1 isolates has a marked effect on antibody association rates and affinities. We also report cryo-EM reconstructions of the apex-targeting PGT145 bnAb bound to two divergent Env that exhibit different degrees of structural dynamics throughout the trimer structures. Parallel HDX-MS experiments demonstrate that PGT145 bnAb has an exquisitely focused footprint at the trimer apex where binding did not yield allosteric changes throughout the rest of the structure. These results demonstrate that structural dynamics are a cryptic determinant of antigenicity, and mature antibodies that have achieved breadth and potency in some cases are able to achieve their broad cross-reactivity by “threading the needle” and binding in a highly focused fashion, thus evading and overcoming the variable properties found in Env from divergent isolates.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

B-294 Delineating the Diversity of Recombinant Monoclonal Antibodies Isolated From Alpaca Polyclonal Mixture

Abstract Introduction Polyclonal antibodies are a mixture of antibodies produced by plasma B-cells and are often obtained by injecting a specific antigen into a host system. They are commonly used for research and diagnostic purposes and are inexpensive and relatively quick to produce. But they lack batch-to-batch reproducibility making them less reliable than recombinant monoclonal antibodies (mAbs). Our team designed a polyclonal sequencing method to sequence the mAbs directly from the immunoserum and then used recombinant technology to reliably express the mAbs. The diversity of the epitopes of these recombinant mAbs are then examined with Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS). Methods In this study, we used a novel mass-spectrometry-based approach to do de novo sequencing of the Alpaca IgGs directly from the immunoserum without analyzing the RNA-seq data. Using our established REpAb sequencing platform and a machine learning-based bioinformatic analysis, we successfully compiled complete amino acid sequences of individual alpaca IgG clones found in the original polyclonal mixture. Using this approach, we isolated ten unique sequences from the polyclonal mixture and expressed them recombinantly. In addition, we performed Hydrogen-deuterium exchange Mass spectrometry experiments on 4 of the ten mAbs to identify their binding sites. For HDX analysis, the antigen digestion was performed using Pepsin/protease XIII column, and the peptides were identified using Waters SELECT SERIES Cyclic IMS Mass spectrometer. Preliminary data The four mAbs which showed strong binding to the antigen were chosen to characterize further using HDX-MS experiments. The preliminary MS/MS experiments provided us with approximately 90% of the amino acid sequence coverage of the antigen. Interestingly, the HDX-MS results showed that different mAbs bind to different antigen regions, which is evident from the deuterium exchange. Overall, our results showed that the monoclonal antibodies isolated from the polyclonal mixture bind to different antigen regions and can represent the diversity in the polyclonal mixture. Novel aspect De novo sequencing functional mAbs from immunoserum and studying their epitope diversities with HDX-MS may help discover novel therapeutics antibodies.

A Functional Bayesian Model for Hydrogen-Deuterium Exchange Mass Spectrometry.

Proteins often undergo structural perturbations upon binding to other proteins or ligands or when they are subjected to environmental changes. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be used to explore conformational changes in proteins by examining differences in the rate of deuterium incorporation in different contexts. To determine deuterium incorporation rates, HDX-MS measurements are typically made over a time course. Recently introduced methods show that incorporating the temporal dimension into the statistical analysis improves power and interpretation. However, these approaches have technical assumptions that hinder their flexibility. Here, we propose a more flexible methodology by reframing these methods in a Bayesian framework. Our proposed framework has improved algorithmic stability, allows us to perform uncertainty quantification, and can calculate statistical quantities that are inaccessible to other approaches. We demonstrate the general applicability of the method by showing it can perform rigorous model selection on a spike-in HDX-MS experiment, improved interpretation in an epitope mapping experiment, and increased sensitivity in a small molecule case-study. Bayesian analysis of an HDX experiment with an antibody dimer bound to an E3 ubiquitin ligase identifies at least two interaction interfaces where previous methods obtained confounding results due to the complexities of conformational changes on binding. Our findings are consistent with the cocrystal structure of these proteins, demonstrating a bayesian approach can identify important binding epitopes from HDX data. We also generate HDX-MS data of the bromodomain-containing protein BRD4 in complex with GSK1210151A to demonstrate the increased sensitivity of adopting a Bayesian approach.

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.

The molecular basis for dysfunctional bacterial lipopolysaccharide-mediated immune receptor activation by SARS-CoV-2 spike.

SARS-CoV-2 has caused hundreds of millions of COVID-19 infections worldwide. While COVID-19 presents with various clinical manifestations, severe COVID-19 disease causes dysregulated host immune reactions that trigger onset of sepsis and acute respiratory distress syndrome. Accumulating evidence suggests that lipopolysaccharide (LPS) derived from the outer membranes of Gram-negative bacteria plays an instrumental role in the progression of such inflammatory states. Patients with pre-existing conditions such as diabetes, hypertension, and obesity are at higher risk to develop severe COVID-19 disease and tend to have significantly elevated blood LPS levels. LPS serves as a signal of bacterial infection upon recognition by Toll-like receptor (TLR4) of the innate immune system, but this can also result in over-amplified immune reactions and sepsis. Here, we present the results of molecular simulations and free-energy calculations, supported by in vitro and in vivo assays and hydrogen-deuterium exchange mass spectrometry experiments, which reveal the molecular mechanism by which the envelope spike glycoprotein of SARS-CoV-2 augments hyperinflammation by acting as a conduit in the TLR4 pathway. LPS was found to bind to several conserved pockets on spike across S1 and S2 subunits. S1 affinity for LPS was comparable to that of CD14, a co-receptor used by immune cells to transfer LPS to TLR4. Cell-based assays and reporter mice experiments showed that low concentrations of spike and LPS synergistically induce a strong pro-inflammatory response, thus pinpointing spike's capacity to “boost” innate immune activation. Finally, the loss of a high-affinity binding site in the Omicron spike led to a reduction of its “boosting” capacity, which may translate to the less severe inflammation observed in patients infected with this variant. Collectively, our findings highlight the potential impact of elevated LPS levels and Gram-negative bacterial coinfections in severe COVID-19 complications.

(-)-Epigallocatechin Gallate is a Noncompetitive Inhibitor of NAD Kinase.

Nicotinamide adenine dinucleotide kinase (NADK) controls the intracellular NADPH content and provides reducing power for the synthesis of macromolecules and anti-ROS. Moreover, NADK is considered to be a synthetic lethal gene for KRAS mutations. To discover NADK-targeted probes, a high-throughput screening assay was established and optimized with a Z factor of 0.71. The natural product (-)-epigallocatechin gallate (EGCG) was found to be a noncompetitive inhibitor of NADK with K i = 3.28 ± 0.32 μΜ. The direct binding of EGCG to NADK was determined by several biophysical methods, including NMR spectroscopy, surface plasmon resonance (SPR) assay, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The SPR assay showed a K d of 1.78 ± 1.15 μΜ. The HDX-MS experiment showed that EGCG was bound at the non-substrate-binding sites of NADK. Besides, binding mode prediction and derivative activity analysis revealed a potential structure-activity relationship between EGCG and NADK. Furthermore, EGCG can specifically inhibit the proliferation of KRAS-mutated lung cancer cell lines without affecting KRAS wild-type lung cancer cell lines.

Simple and Fast Maximally Deuterated Control (maxD) Preparation for Hydrogen-Deuterium Exchange Mass Spectrometry Experiments.

During the analysis steps of hydrogen-deuterium exchange (HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during liquid chromatography (LC)-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD), in which the protein is deuterated at positions and analyzed with the same buffer components, %D2O, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here, we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ∼40 min for nonmembrane proteins. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with earlier protocols that either require much more time or require isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed for maximum deuteration in a short time (∼10 min) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy-to-perform protocol should enhance routine preparation of maxD controls.

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

The COVID-19 pandemic has prompted a rapid response in vaccine and drug development. Herein, we modeled a complete membrane-embedded SARS-CoV-2 spike glycoprotein and used molecular dynamics simulations with benzene probes designed to enhance discovery of cryptic pockets. This approach recapitulated lipid and host metabolite binding sites previously characterized by cryo-electron microscopy, revealing likely ligand entry routes, and uncovered a novel cryptic pocket with promising druggable properties located underneath the 617–628 loop. A full representation of glycan moieties was essential to accurately describe pocket dynamics. A multi-conformational behavior of the 617–628 loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry experiments, supportive of opening and closing dynamics. The pocket is the site of multiple mutations associated with increased transmissibility found in SARS-CoV-2 variants of concern including Omicron. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

Probing the Impact of Temperature and Substrates on the Conformational Dynamics of the Neurotransmitter:Sodium symporter LeuT

The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure–function relationships of NSS family proteins. With hydrogen–deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 – 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against Neisseria meningitidis serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.

Hydrogen-Deuterium Exchange Mass Spectrometry for Probing Changes in Conformation and Dynamics of Proteins.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is, nowadays, an increasingly important technique in studying protein conformation and dynamics. This technique possesses the advantages of low sample consumption, less limitation in protein size, and relatively simple experimental workflow. An HDX-MS experiment typically includes the steps of sample preparation, HDX reaction, quenching of HDX reaction, protease digestion, and LC-MS analysis. Although HDX-MS has been an established technique and automatic sample handling devices are commercially available nowadays, proper experimental conditions of each step are crucial for a successful HDX-MS experiment. This chapter is to provide a general guideline for each step in the HDX-MS workflow and highlight some precautions needed to be taken in order to acquire useful conformational and dynamic information.

HDX-MS: An Analytical Tool to Capture Protein Motion in Action.

Virtually all protein functions in the cell, including pathogenic processes, require coordinated motion of atoms or domains, i.e., conformational dynamics. Understanding protein dynamics is therefore critical both for drug development and to learn about the underlying molecular causes of many diseases. Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) provides valuable information about protein dynamics, which is highly complementary to the static picture provided by conventional high-resolution structural tools (i.e., X-ray crystallography and structural NMR). The amount of protein required to carry out HDX-MS experiments is a fraction of the amount required by alternative biophysical techniques, which are also usually lower resolution. Use of HDX-MS is growing quickly both in industry and academia, and it has been successfully used in numerous drug and vaccine development efforts, with important roles in understanding allosteric effects and mapping binding sites.

Dynamics of Hepatitis B Virus Capsid Protein Dimer Regulate Assembly through an Allosteric Network.

While there is an effective vaccine for Human Hepatitis B Virus (HBV), 257 million people have chronic infections for which there is no cure. The assembly process for the viral capsid is a potential therapeutic target. In order to understand the capsid assembly process, we investigated the dimeric building blocks of the capsid. To understand what blocks assembly, we took advantage of an assembly incompetent mutant dimer, Cp149-Y132A, located in the interdimer interface. This mutation leads to changes in protein dynamics throughout the structure of the dimer as measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS). To further understand how the HBV capsid assembles, the homologue woodchuck HBV (WHV) capsid protein dimer (Cp) was used. WHV is more stable than HBV in HDX-MS and native mass spectrometry experiments. Because the WHV Cp assembles more rapidly into viral capsids than HBV, it was suspected that an increase in stability of the intradimer interface and/or in the contact region leads to increased assembly rates. The differences in dynamics when comparing HBV and human Cp149-Y132A as well as the differences in dynamics when comparing the HBV and WHV Cps allowed us to map an allosteric network within the HBV dimer. Through a careful comparison of structure, stability, and dynamics using four different capsid protein dimers, we conclude that protein subunit dynamics regulate HBV capsid assembly.

HaDeX: an R package and web-server for analysis of data from hydrogen-deuterium exchange mass spectrometry experiments.

MotivationHydrogen–deuterium mass spectrometry (HDX-MS) is a rapidly developing technique for monitoring dynamics and interactions of proteins. The development of new devices has to be followed with new software suites addressing emerging standards in data analysis.ResultsWe propose HaDeX, a novel tool for processing, analysis and visualization of HDX-MS experiments. HaDeX supports a reproducible analytical process, including data exploration, quality control and generation of publication-quality figures.Availability and implementationHaDeX is available primarily as a web-server (http://mslab-ibb.pl/shiny/HaDeX/), but its all functionalities are also accessible as the R package (https://CRAN.R-project.org/package=HaDeX) and standalone software (https://sourceforge.net/projects/HaDeX/).Supplementary information Supplementary data are available at Bioinformatics online.

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.