Publications by authors named "Sébastien Brier"

33 Publications

A High-Affinity Calmodulin-Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells.

Adv Sci (Weinh) 2021 05 8;8(9):2003630. Epub 2021 Mar 8.

Biochemistry of Macromolecular Interactions Unit Department of Structural Biology and Chemistry Institut Pasteur CNRS UMR3528 Paris 75015 France.

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 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.
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http://dx.doi.org/10.1002/advs.202003630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097335PMC
May 2021

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

FASEB J 2021 May;35(5):e21540

Institut Pasteur, Unité des Toxines Bactériennes, UMR CNRS 2001, Paris, France.

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 H and H subdomains of the BoNT/A1 receptor-binding domain (H ). 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 H 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.
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http://dx.doi.org/10.1096/fj.202002492RDOI Listing
May 2021

Hydrogen/Deuterium Exchange Mass Spectrometry for the Structural Analysis of Detergent-Solubilized Membrane Proteins.

Methods Mol Biol 2020 ;2127:339-358

Biological NMR Technological Platform, Center for Technological Resources and Research, Department of Structural Biology and Chemistry, Institut Pasteur, CNRS UMR3528, Paris, France.

Integral membrane proteins are involved in numerous biological functions and represent important drug targets. Despite their abundance in the human proteome, the number of integral membrane protein structures is largely underrepresented in the Protein Data Bank. The challenges associated with the biophysical characterization of such biological systems are well known. Most structural approaches, including X-ray crystallography, SAXS, or mass spectrometry (MS), require the complete solubilization of membrane proteins in aqueous solutions. Detergents are frequently used for this task, but may interfere with the analysis, as is the case with MS. The use of "MS-friendly" detergents, such as non-ionic alkyl glycoside detergents, has greatly facilitated the analysis of detergent-solubilized membrane proteins. Here, we describe a protocol, which we have successfully implemented in our laboratory to study the structure and dynamics of detergent-solubilized integral membrane proteins by Hydrogen/Deuterium eXchange and Mass Spectrometry (HDX-MS). The procedure does not require detergent removal prior to MS analysis, instead taking advantage of the ultra-high pressure chromatographic system to separate deuterated peptides from "MS-friendly" detergents.
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http://dx.doi.org/10.1007/978-1-0716-0373-4_22DOI Listing
March 2021

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

Nat Methods 2019 07 27;16(7):595-602. Epub 2019 Jun 27.

Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.

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.
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http://dx.doi.org/10.1038/s41592-019-0459-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614034PMC
July 2019

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

FASEB J 2019 09 21;33(9):10065-10076. Epub 2019 Jun 21.

Institut Pasteur, Chemistry and Structural Biology Department, UMR CNRS 3528, Paris, France.

The adenylate cyclase (CyaA) toxin is a major virulence factor of , 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.
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http://dx.doi.org/10.1096/fj.201802442RRDOI Listing
September 2019

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

J Biomol NMR 2019 Jul 23;73(6-7):293-303. Epub 2019 May 23.

Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, C3BI, Institut Pasteur; CNRS UMR3528; CNRS USR3756, Paris, France.

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.
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http://dx.doi.org/10.1007/s10858-019-00246-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692295PMC
July 2019

Translocation and calmodulin-activation of the adenylate cyclase toxin (CyaA) of Bordetella pertussis.

Pathog Dis 2018 11 1;76(8). Epub 2018 Nov 1.

Département de Biologie Structurale et Chimie, Institut Pasteur, CNRS UMR 3528, 28 Rue du Dr Roux, 75724 Paris, CEDEX 15, France.

The adenylate cyclase toxin (CyaA) is a multi-domain protein secreted by Bordetella pertussis, the causative agent of whooping cough. CyaA is involved in the early stages of respiratory tract colonization by Bordetella pertussis. CyaA is produced and acylated in the bacteria, and secreted via a dedicated secretion system. The cell intoxication process involves a unique mechanism of transport of the CyaA toxin catalytic domain (ACD) across the plasma membrane of eukaryotic cells. Once translocated, ACD binds to and is activated by calmodulin and produces high amounts of cAMP, subverting the physiology of eukaryotic cells. Here, we review our work on the identification and characterization of a critical region of CyaA, the translocation region, required to deliver ACD into the cytosol of target cells. The translocation region contains a segment that exhibits membrane-active properties, i.e. is able to fold upon membrane interaction and permeabilize lipid bilayers. We proposed that this region is required to locally destabilize the membrane, decreasing the energy required for ACD translocation. To further study the translocation process, we developed a tethered bilayer lipid membrane (tBLM) design that recapitulate the ACD transport across a membrane separating two hermetic compartments. We showed that ACD translocation is critically dependent on calcium, membrane potential, CyaA acylation and on the presence of calmodulin in the trans compartment. Finally, we describe how calmodulin-binding triggers key conformational changes in ACD, leading to its activation and production of supraphysiological concentrations of cAMP.
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http://dx.doi.org/10.1093/femspd/fty085DOI Listing
November 2018

Consensus designs and thermal stability determinants of a human glutamate transporter.

Elife 2018 10 18;7. Epub 2018 Oct 18.

Molecular Mechanisms of Membrane Transport Laboratory, Institut Pasteur, Paris, France.

Human 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.
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http://dx.doi.org/10.7554/eLife.40110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209432PMC
October 2018

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough.

Toxicon 2018 Jul 12;149:37-44. Epub 2018 Jan 12.

Institut Pasteur, Biochemistry of Macromolecular Interactions Unit, UMR CNRS 3528, Structural Biology and Chemistry Department, 75724 PARIS Cedex 15, France. Electronic address:

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.
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http://dx.doi.org/10.1016/j.toxicon.2018.01.007DOI Listing
July 2018

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.

PLoS Biol 2017 12 29;15(12):e2004486. Epub 2017 Dec 29.

Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, Paris, France.

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.
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http://dx.doi.org/10.1371/journal.pbio.2004486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764468PMC
December 2017

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

Biochem J 2018 01 15;475(1):341-354. Epub 2018 Jan 15.

Laboratoire Systèmes Macromoléculaires et Signalisation, Département de Microbiologie, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France

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/σ 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 σ 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 σ Instead, we show that σ adopts an open conformation in solution in which the folded σ and σ 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 σ and provide insights into the possible mechanisms of regulation of σ activity by its chaperone Crl.
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http://dx.doi.org/10.1042/BCJ20170768DOI Listing
January 2018

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

Sci Rep 2017 Nov 14;7(1):15544. Epub 2017 Nov 14.

Aix-Marseille Univ, CNRS, Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257, Marseille, France.

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).
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http://dx.doi.org/10.1038/s41598-017-15299-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686140PMC
November 2017

Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem.

Structure 2017 11 28;25(11):1645-1656.e5. Epub 2017 Sep 28.

CNRS, UMR 3528, 75015 Paris, France; Unité de Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur, 75015 Paris, France. Electronic address:

Hearing relies on the transduction of sound-evoked vibrations into electric signals, occurring in the stereocilia bundle of hair cells. The bundle is organized in a staircase pattern formed by rows of packed stereocilia. This architecture is pivotal to transduction and involves a network of scaffolding proteins with hitherto uncharacterized features. Key interactions in this network are mediated by PDZ domains. Here, we describe the architecture of the first two PDZ domains of whirlin, a protein involved in these assemblies and associated with congenital deaf-blindness. C-terminal hairpin extensions of the PDZ domains mediate the transient supramodular assembly, which improves the binding capacity of the first domain. We determined a detailed structural model of the closed conformation of the PDZ tandem and characterized its equilibrium with an ensemble of open conformations. The structural and dynamic behavior of this PDZ tandem provides key insights into the regulatory mechanisms involved in the hearing machinery.
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http://dx.doi.org/10.1016/j.str.2017.08.013DOI Listing
November 2017

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

Biotechnol Appl Biochem 2018 Jan 2;65(1):62-68. Epub 2017 Oct 2.

Institut de Biologie Intégrative de la Cellule, UMR 9198, Université Paris-Sud, Orsay, France.

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.
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http://dx.doi.org/10.1002/bab.1577DOI Listing
January 2018

Structure and allosteric inhibition of excitatory amino acid transporter 1.

Nature 2017 04 19;544(7651):446-451. Epub 2017 Apr 19.

Molecular Mechanisms of Membrane Transport Laboratory, Institut Pasteur, 25-28 rue du Docteur Roux, 75015 Paris, France.

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.
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http://dx.doi.org/10.1038/nature22064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410168PMC
April 2017

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

J Biol Chem 2017 Jan 30;292(1):328-338. Epub 2016 Nov 30.

From the Molecular Genetics Unit, CNRS ERL 3526,

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 N 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 N domain stability, suggested that the N 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 β-barrel assembly machinery.
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http://dx.doi.org/10.1074/jbc.M116.759498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217691PMC
January 2017

Proteomics for Allergy: from Proteins to the Patients.

Curr Allergy Asthma Rep 2016 09;16(9):64

Stallergenes Greer, Research Department, 6 rue Alexis de Tocqueville, 92183, Antony cedex, France.

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.
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http://dx.doi.org/10.1007/s11882-016-0642-5DOI Listing
September 2016

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

Bioinformatics 2016 11 13;32(22):3413-3419. Epub 2016 Jul 13.

Department of Chemistry and Structural Biology, Institut Pasteur, UMR CNRS, Paris 3528, France.

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: [email protected] or [email protected] information: Supplementary data is available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btw420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181559PMC
November 2016

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

PLoS Pathog 2016 04 22;12(4):e1005557. Epub 2016 Apr 22.

GSK Vaccines Srl, Siena, Italy.

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.
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http://dx.doi.org/10.1371/journal.ppat.1005557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841544PMC
April 2016

Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion.

Sci Rep 2015 Sep 16;5:14223. Epub 2015 Sep 16.

Institut Pasteur, UMR CNRS 3528, Chemistry and Structural Biology Department, 75724 PARIS cedex 15, France.

Many Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound Holo-state. Apo-RD behaves as a disordered polymer chain comprising several statistical elements that exhibit local rigidity with residual secondary structure. Holo-RD is a folded multi-domain protein with an anisometric shape. RTX motifs thus appear remarkably adapted to the structural and mechanistic constraints of the secretion process. In the low calcium environment of the bacterial cytosol, Apo-RD is an elongated disordered coil appropriately sized for transport through the narrow secretion machinery. The progressive folding of Holo-RD in the extracellular calcium-rich environment as it emerges form the T1SS may then favor its unidirectional export through the secretory channel. This process is relevant for hundreds of bacterial species producing virulent RTX proteins.
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http://dx.doi.org/10.1038/srep14223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642704PMC
September 2015

Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

FASEB J 2015 Jun 20;29(6):2260-7. Epub 2015 Feb 20.

Novartis Vaccines, Siena, Italy

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).
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http://dx.doi.org/10.1096/fj.14-263954DOI Listing
June 2015

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

FASEB J 2014 Apr 26;28(4):1644-53. Epub 2013 Dec 26.

1Research Center, Novartis Vaccines and Diagnostics Srl, Via Fiorentina 1, 53100 Siena, Italy.

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.
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http://dx.doi.org/10.1096/fj.13-239012DOI Listing
April 2014

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

Proc Natl Acad Sci U S A 2013 Feb 8;110(9):3304-9. Epub 2013 Feb 8.

Research Center, Novartis Vaccines and Diagnostics srl, 53100 Siena, Italy.

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.
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http://dx.doi.org/10.1073/pnas.1222845110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587270PMC
February 2013

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

Infect Immun 2013 Feb 10;81(2):560-9. Epub 2012 Dec 10.

Novartis Vaccines and Diagnotics, Research Center, Siena, Italy.

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.
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http://dx.doi.org/10.1128/IAI.01085-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553804PMC
February 2013

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

Biochemistry 2012 Aug 15;51(34):6738-52. Epub 2012 Aug 15.

Research Center, Novartis Vaccines and Diagnostics, via Fiorentina 1, 53100, Siena, Italy.

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.
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http://dx.doi.org/10.1021/bi300656wDOI Listing
August 2012

Mapping surface accessibility of the C1r/C1s tetramer by chemical modification and mass spectrometry provides new insights into assembly of the human C1 complex.

J Biol Chem 2010 Oct 30;285(42):32251-63. Epub 2010 Jun 30.

CNRS, UMR 8587, Université d'Evry-Val-d'Essonne, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Boulevard François Mitterrand, 91025 Evry, France.

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.
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http://dx.doi.org/10.1074/jbc.M110.149112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952226PMC
October 2010

Aspartic proteinases in Antarctic fish.

Mar Genomics 2009 Mar 1;2(1):1-10. Epub 2009 Apr 1.

CNR, Institute of Protein Biochemistry, Via P. Castellino 111-80131, Naples, Italy.

The present review surveys several recent studies of the aspartic proteinases from Antarctic Notothenioidei, a dominating fish group that has developed a number of adjustments at the molecular level to maintain metabolic function at low temperatures. Given the unique peculiarities of the Antarctic environment, studying the features of Antarctic aspartic proteinases could provide new insights into the role of these proteins in fish physiology. We describe here: (1) the biochemical properties of a cathepsin D purified from the liver of the hemoglobinless icefish Chionodraco hamatus; (2) the biochemical characterization of Trematomus bernacchii pepsins variants A1 and A2 obtained by heterologous expression in bacteria; and (3) the identification of two closely related, novel aspartic proteinases from the liver of the two Antarctic fish species mentioned above. Overall, the results show that Notothenioidei aspartic proteinases display a number of characteristics that are remarkably different from those of mammalian aspartic proteinases, including high turnover number or high catalytic efficiency. We have named the newly identified aspartic proteinases "Nothepsins" and classified them relative to aspartic proteinases from other species.
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http://dx.doi.org/10.1016/j.margen.2009.03.001DOI Listing
March 2009

Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod Trematomus bernacchii.

FEBS J 2007 Dec 1;274(23):6152-66. Epub 2007 Nov 1.

Department of Chemistry & Chemical Biology and The Barnett Institute for Chemical & Biological Analysis, Northeastern University, Boston, MA, USA.

The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.
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http://dx.doi.org/10.1111/j.1742-4658.2007.06136.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533623PMC
December 2007

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

Protein Sci 2007 Apr 27;16(4):572-81. Epub 2007 Feb 27.

Chemistry and Chemical Biology, The Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115, USA.

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.
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http://dx.doi.org/10.1110/ps.062631007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2203333PMC
April 2007

The marine natural product adociasulfate-2 as a tool to identify the MT-binding region of kinesins.

Biochemistry 2006 Dec 1;45(51):15644-53. Epub 2006 Dec 1.

Laboratoire de Spectrométrie de Masse des Protéines (LSMP), Institut de Biologie Structurale (CEA-CNRS-UJF), 41, rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

Kinesins are molecular motors that transport cargo along microtubules (MTs). To move forward the motor must attach to the MT in a defined orientation and detach from it in a process that is driven by ATP hydrolysis. The knowledge of the motor-MT interface is essential for a detailed understanding of how kinesins move along MTs and how they are related to other molecular motors such as myosins or dyneins. We have used the marine natural product adociasulfate-2 (AS-2), previously identified as a MT-competitive inhibitor of conventional kinesin, to infer the secondary structure elements forming the MT interface of two human mitotic kinesins, namely, CENP-E and Eg5. AS-2 inhibits both basal and MT-stimulated ATPase activities of CENP-E (IC50 of 8.6 and 1.3 microM, respectively) and Eg5 (IC50 of 3.5 and 5.3 microM, respectively) and is a MT-competitive inhibitor of CENP-E with a Ki of 0.35 microM. Binding of AS-2 to CENP-E also stimulates the ADP release from the nucleotide-binding pocket. AS-2 is a nonspecific kinesin inhibitor targeting several superfamily members including KHC, MPP1, MKLP1, RabK6, KIFC1, KIFC3, CENP-E, and Eg5. By measuring hydrogen/deuterium exchange with mass spectrometry we have shown that the formation of the CENP-E/AS-2 complex decreases the solvent accessibility of three neighboring peptides on the same face of CENP-E. We deduce that this is the site of MT attachment and conclude that loop L11, helix alpha4, loop L12, helix alpha5, loop L8, and strand beta5 constitute the main MT interface of the CENP-E motor domain. Similarly for Eg5/AS-2, a region of increased solvent accessibility locates the MT interface of Eg5.
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http://dx.doi.org/10.1021/bi061395nDOI Listing
December 2006