A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion of eukaryotic cells

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454-484), which exhibits membrane-active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells

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

Etude des interactions entre la kinésine mitotique humaine Eg5 et ses inhibiteurs

La kinésine mitotique humaine HsEg5 est essentielle à la division cellulaire. Ce moteur moléculaire permet la séparation des centrosomes et la mise en place du fuseau mitotique, structure nécessaire au partage équitable de l'information génétique. La suppression de la fonction d'HsEg5 bloque les cellules en pré-métaphase avec un fuseau mitotique monoastral caractéristique constitué des deux centrosomes non séparés entourés d'un anneau de chromosomes et de microtubules. Le maintien de ce phénotype peut conduire à la mort cellulaire programmée via l'activation du point de contrôle du fuseau mitotique (transition métaphase-anaphase). HsEg5 est ainsi considérée comme une cible anticancéreuse particulièrement intéressante. Au cours de ces travaux, nous nous sommes intéressés aux interactions entre le domaine moteur d'HsEg5 et plusieurs inhibiteurs. Les zones d'interaction et de modifications conformationnelles ont été étudiées par échanges hydrogène/deutérium spectrométrie de masse et mutagenèse dirigée. Cette approche expérimentale nous a permis d'identifier un " point chaud " d'inhibition sur le domaine moteur et de caractériser les mécanismes de deux inhibiteurs : le monastrol et le S-trityl-l-cystéine.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

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

International audienceSecretion 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

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

International audienceCompared 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

Structural disorder and induced folding within two cereal, ABA stress and ripening (ASR) proteins

Abstract Abscisic acid (ABA), stress and ripening (ASR) proteins are plant-specific proteins involved in plant response to multiple abiotic stresses. We previously isolated the ASR genes and cDNAs from durum wheat (TtASR1) and barley (HvASR1). Here, we show that HvASR1 and TtASR1 are consistently predicted to be disordered and further confirm this experimentally. Addition of glycerol, which mimics dehydration, triggers a gain of structure in both proteins. Limited proteolysis showed that they are highly sensitive to protease degradation. Addition of 2,2,2-trifluoroethanol (TFE) however, results in a decreased susceptibility to proteolysis that is paralleled by a gain of structure. Mass spectrometry analyses (MS) led to the identification of a protein fragment resistant to proteolysis. Addition of zinc also induces a gain of structure and Hydrogen/Deuterium eXchange-Mass Spectrometry (HDX-MS) allowed identification of the region involved in the disorder-to-order transition. This study is the first reported experimental characterization of HvASR1 and TtASR1 proteins, and paves the way for future studies aimed at unveiling the functional impact of the structural transitions that these proteins undergo in the presence of zinc and at achieving atomic-resolution conformational ensemble description of these two plant intrinsically disordered proteins (IDPs)

Consensus designs and thermal stability determinants of a human glutamate transporter

International audienceHuman excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs' molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~95% amino acid identity with the wild type transporters, and remain natively folded and functional. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses

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

International audienceMotivation: 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 ∆D " , 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: MEMHDX is freely available as a web tool at the project home page http://memhdx