Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

Structure and allosteric inhibition of excitatory amino acid transporter 1.

Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is associated with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the molecular mechanisms underlying the function and pharmacology of human SLC1 transporters.

Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium.

BACKGROUND: Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. RESULTS: We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. CONCLUSION: Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A.

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the HCN and HCC subdomains of the BoNT/A1 receptor-binding domain (HC ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to HC confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.

Post-translational acylation controls the folding and functions of the CyaA RTX toxin.

The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro-CyaA and CyaA proteins. HDX-MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C-terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro-CyaA. Once refolded into monomers, CyaA is more compact and stable than pro-CyaA, due to a complex set of interactions between domains. Our HDX-MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation-dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post-translational modifications, structural disorder and protein folding. Coupling calcium-binding and acylation-driven folding is likely pertinent for other repeat-in-toxin cytolysins produced by many Gram-negative bacterial pathogens.-O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux-Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post-translational acylation controls the folding and functions of the CyaA RTX toxin.

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes.

Dynamics of a type 2 secretion system pseudopilus unraveled by complementary approaches.

Secretion pili, bacterial fibers responsible for transporting proteins to the extracellular milieu in some secretion systems, are very strong structures but at the same time highly flexible. Their flexibility and helical symmetry make structure determination at atomic resolution a challenging task. We have previously used an integrative structural biology approach including liquid-state NMR, cryo-electron microscopy (cryo-EM), and modeling to determine the pseudo-atomic resolution structure of the type 2 secretion system pseudopilus in a mutant form, where we employed NMR to determine the high resolution structure of the pilin (the monomer building block of the pilus). In this work, we determine the pseudo-atomic structure of the wild type pilus, and compare the dynamics of wild type and mutant pili by normal mode analysis. We present a detailed NMR analysis of the dynamics of the pilin in isolation, and compare dynamics and solvent accessibility of isolated and assembled pilins by Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS). These complementary approaches provide a comprehensive view of internal and overall dynamics of pili, crucial for their function.

The stress sigma factor of RNA polymerase RpoS/σS is a solvent-exposed open molecule in solution.

In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.

Prepore Stability Controls Productive Folding of the BAM-independent Multimeric Outer Membrane Secretin PulD.

Members of a group of multimeric secretion pores that assemble independently of any known membrane-embedded insertase in Gram-negative bacteria fold into a prepore before membrane-insertion occurs. The mechanisms and the energetics that drive the folding of these proteins are poorly understood. Here, equilibrium unfolding and hydrogen/deuterium exchange monitored by mass spectrometry indicated that a loss of 4-5 kJ/mol/protomer in the N3 domain that is peripheral to the membrane-spanning C domain in the dodecameric secretin PulD, the founding member of this class, prevents pore formation by destabilizing the prepore into a poorly structured dodecamer as visualized by electron microscopy. Formation of native PulD-multimers by mixing protomers that differ in N3 domain stability, suggested that the N3 domain forms a thermodynamic seal onto the prepore. This highlights the role of modest free energy changes in the folding of pre-integration forms of a hyperstable outer membrane complex and reveals a key driving force for assembly independently of the ß-barrel assembly machinery.

Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.

SEC-SAXS and HDX-MS: A powerful combination. The case of the calcium-binding domain of a bacterial toxin.

Small-angle X-ray scattering (SAXS) is a relatively simple experimental technique that provides information on the global conformation of macromolecules in solution, be they fully structured, partially, or extensively unfolded. Size exclusion chromatography in line with a SAXS measuring cell considerably improves the monodispersity and ideality of solutions, the two main requirements of a "good" SAXS sample. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) offers a wealth of information regarding the solvent accessibility at the local (peptide) level. It constitutes a sensitive probe of local flexibility and, more generally, of structural dynamics. The combination of both approaches presented here is very powerful, as illustrated by the case of RD, a calcium-binding protein that is part of a bacterial virulence factor.

MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets.

MOTIVATION: With the continued improvement of requisite mass spectrometers and UHPLC systems, Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) workflows are rapidly evolving towards the investigation of more challenging biological systems, including large protein complexes and membrane proteins. The analysis of such extensive systems results in very large HDX-MS datasets for which specific analysis tools are required to speed up data validation and interpretation. RESULTS: We introduce a web application and a new R-package named 'MEMHDX' to help users analyze, validate and visualize large HDX-MS datasets. MEMHDX is composed of two elements. A statistical tool aids in the validation of the results by applying a mixed-effects model for each peptide, in each experimental condition, and at each time point, taking into account the time dependency of the HDX reaction and number of independent replicates. Two adjusted P-values are generated per peptide, one for the 'Change in dynamics' and one for the 'Magnitude of ΔD', and are used to classify the data by means of a 'Logit' representation. A user-friendly interface developed with Shiny by RStudio facilitates the use of the package. This interactive tool allows the user to easily and rapidly validate, visualize and compare the relative deuterium incorporation on the amino acid sequence and 3D structure, providing both spatial and temporal information. AVAILABILITY AND IMPLEMENTATION: MEMHDX is freely available as a web tool at the project home page http://memhdx.c3bi.pasteur.fr CONTACT: marie-agnes.dillies@pasteur.fr or sebastien.brier@pasteur.frSupplementary information: Supplementary data is available at Bioinformatics online.

Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

Proteomics for Allergy: from Proteins to the Patients.

Proteomics encompasses a variety of approaches unraveling both the structural features, post-translational modifications, and abundance of proteins. As of today, proteomic studies have shed light on the primary structure of about 850 allergens, enabling the design of microarrays for improved molecular diagnosis. Proteomic methods including mass spectrometry allow as well to investigate protein-protein interactions, thus yielding precise information on critical epitopes on the surface of allergens. Mass spectrometry is now being applied to the unambiguous identification, characterization, and comprehensive quantification of allergens in a variety of matrices, as diverse as food samples and allergen immunotherapy drug products. As such, it represents a method of choice for quality testing of allergen immunotherapy products.

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.

Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes.

Transcriptional regulation of the nadA gene in Neisseria meningitidis impacts the prediction of coverage of a multicomponent meningococcal serogroup B vaccine.

The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B (MenB) infection. Under in vitro growth conditions, nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killing of MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadA in laboratory growth nevertheless recognize the NadA antigen, suggesting that NadA expression during infection may be different from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR. All nadR knockout strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with 4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infants when the strain is grown in vitro. However, in an in vivo passive protection model, the same sera effectively protected infant rats from bacteremia with NGP165. Furthermore, we identify a novel hydroxyphenylacetic acid (HPA) derivative, reported by others to be produced during inflammation, which induces expression of NadA in vitro, leading to efficient antibody-mediated killing. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h postinfection. Our results suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levels of NadA expression from any nadA-positive (nadA(+)) strain and therefore efficient killing by anti-NadA antibodies elicited by the 4CMenB vaccine.

Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system.

Type II secretion systems (T2SSs) allow diderm bacteria to secrete hydrolytic enzymes, adhesins, or toxins important for growth and virulence. To promote secretion of folded proteins, T2SSs assemble periplasmic filaments called pseudopili or endopili at an inner membrane subcomplex, the assembly platform (AP). Here, we combined biophysical approaches, nuclear magnetic resonance (NMR) and X-ray crystallography, to study the Klebsiella AP components PulL and PulM. We determined the structure and associations of their periplasmic domains and describe the structure of the heterodimer formed by their ferredoxin-like domains. We show how structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and crosslinking data provided additional constraints to build a structural model of the PulL-PulM assembly in the cellular context. Our structural and functional insights, together with the relative cellular abundance of its components, support the role of AP as a dynamic hub that orchestrates pilus polymerization.

Structural insight into the mechanism of DNA-binding attenuation of the Neisserial adhesin repressor NadR by the small natural ligand 4-hydroxyphenylacetic acid.

Neisserial adhesin A (NadA) is a surface exposed trimeric protein present in most hypervirulent meningococcal strains and involved in epithelial cell adhesion and colonization. The expression of nadA is controlled by Neisserial adhesin regulator (NadR), a member of the MarR family, which binds to the nadA promoter and strongly represses the transcription of nadA. It was recently demonstrated that the DNA-binding activity of NadR was attenuated by 4-hydroxyphenylacetic acid (4-HPA), a natural molecule released in human saliva, thus leading to the de-repression of nadA in vivo. To elucidate the mechanism of regulation of NadR by 4-HPA, we used hydrogen-deuterium exchange mass spectrometry in association with in silico docking and site-directed mutagenesis. We show here that 4-HPA binds at the interface between the dimerization and the DNA-binding domains and stabilizes the homodimeric state of NadR without inducing large conformational changes in the DNA-binding lobes. The residues predicted to be in contact with 4-HPA were further selected for mutagenesis to assess their in vitro and in vivo functions in 4-HPA binding. Our results indicate that Arg(40) is critical for DNA-binding and reveal that Tyr(115) plays a key role in the mechanism of regulation of NadR by 4-HPA. Altogether our data suggest that the mechanism of regulation of NadR by 4-HPA mainly involves the stabilization of the dimer in a configuration incompatible with DNA binding.