Caractérisation structurale de protéines membranaires par échange hydrogène/deutérium spectrométrie de masse

Des exporteurs ATP-Binding Cassette (ABC) sont connus pour être responsables de la résistance contre un large spectre d'agents antibiotiques ou chimiothérapeutiques chez les bactéries et les cellules de mammifères. L'échange hydrogène / deutérium associé à la spectrométrie de masse (HDX-MS) a été utilisé pour caractériser les changements conformationnels de deux exporteurs ABC bactériens, BmrA et BmrC/BmrD, en présence et en absence de nucléotide. Les cinétiques locales d'HDX ont montré la nature hautement dynamique des domaines intra-cellulaires (ICDs) dans la forme apo, ce qui n'était pas attendu d'après les structures cristallographiques aux rayons X des protéines homologues. Dans la conformation ouverte vers l'extérieur ( outward facing ), domaines fixant les nucléotides (NBDs) interagissant, le mouvement des ICDs sont largement réduits pour les deux transporteurs. Les cinétiques d'HDX MS dans la conformation outward facing ont été déterminées en appliquant cette technique sur des mutants incapables d'hydrolyser les nucléotides et sur la forme sauvage inhibée au vanadate. La dynamique des NBDs, en particulier pour les régions qui interagissent au cours de l'hydrolyse de l'ATP, a été aussi diminuée dans la conformation outward facing comparativement à celle ouverte vers l'intérieur. L'ajout de différents agents connus pour être transportés par des transporteurs ABC n'a pas affecté la dynamique des NBDs. Par ailleurs, nous avons aussi appliqué l'HDX MS à la protéine GLIC, un homologue procaryote d'un ligand-gated ion channel pentamérique (pLGIC). Les cinétiques locales d'HDX sont en plein accord avec la structure cristallographique disponible et le changement de pH révèle des différences de deutération dans les régions d'interaction des sous-unités.Some ATP-Binding Cassette exporters are known to be responsible for resistance against a broad spectrum of antibiotics and chemotherapeutic drugs in bacteria and mammalian cells. Amide hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) was applied to investigate the conformational changes in two different bacterial ABC exporters, BmrA and BmrC/BmrD, in the presence and absence of nucleotide. Local H/D exchange kinetics showed highly dynamic nature of ICDs in apo form which was not anticipated from X-ray structure of the homologue proteins. In outward facing (closed form) conformation the movement of ICDs were largely reduced for both transporters. The H/D exchange kinetics of closed form were determined by applying H/D exchange on mutants unable to hydrolyze nucleotides or on wild-type inhibited by vanadate. The dynamics of NBDs particularly for those regions which interact during ATP hydrolysis were also reduced in closed form as compared to open one. The addition of different drugs which are known to be transported by ABC transporters did not affect dynamics of NBDs. We further applied H/D exchange kinetics on a prokaryotic homologue of pentameric ligandgated ion channel (pLGIC) GLIC. Local H/D exchange kinetics were in full agreement with the available structure and change in pH showed differences in deuterium level for interacting regions of the subunits.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Single Nanoparticle Plasmonic Spectroscopy for Study of the Efflux Function of Multidrug ABC Membrance Transporters of Single Live Cells

ATP-binding cassette (ABC) membrane transporters exist in all living organisms and play key roles in a wide range of cellular and physiological functions. The ABC transporters can selectively extrude a wide variety of structurally and functionally unrelated substrates, leading to multidrug resistance. Despite extensive study, their efflux molecular mechanisms remain elusive. In this study, we synthesized and characterized purified silver nanoparticles (Ag NPs) (97 ± 13 nm in diameter), and used them as photostable optical imaging probes to study efflux kinetics of ABC membrane transporters (BmrA) of single live cells (B. subtilis). The NPs with concentrations up to 3.7 pM were stable (non-aggregated) in a PBS buffer and biocompatible with the cells. We found a high dependence of accumulation of the intracellular NPs in single live cells (WT, Ct-BmrAEGFP, ΔBmrA) upon the cellular expression level of BmrA and NP concentration (0.93, 1.85 and 3.7 pM), showing the highest accumulation of intracellular NPs in ΔBmrA (deletion of BmrA) and the lowest ones in Ct-BmrA-EGFP (over-expression of BmrA). Interestingly, the accumulation of intracellular NPs in ΔBmrA increases nearly proportionally with the NP concentration, while those in WT and Ct-BmrA-EGFP do not. This result suggests that the NPs enter the cells via passive diffusion driven by concentration gradients across the cellular membrane and they are extruded out of cells by BmrA transporters, similar to conventional pump substrates (antibiotics). This study shows that such large substrates (84-100 nm NPs) can enter into the live cells and be extruded out of the cells by BmrA, and the NPs can serve as nm-sized optical imaging probes to study the size-dependent efflux kinetics of membrane transporters in single live cells in real time

The Tyrosine-Autokinase UbK Is Required for Proper Cell Growth and Cell Morphology of Streptococcus pneumoniae

International audienceProtein phosphorylation is a key post-translational modification required for many cellular functions of the bacterial cell. Recently, we identified a new protein-kinase, named UbK, in Bacillus subtilis that belongs to a new family of protein-kinases widespread in bacteria. In this study, we analyze the function of UbK in Streptococcus pneumoniae. We show that UbK displays a tyrosine-kinase activity and autophosphorylates on a unique tyrosine in vivo. To get insights into its cellular role, we constructed a set of pneumococcal ubk mutants. Using conventional and electron microscopy, we show that the ubk deficient strain, as well as an ubk catalytic dead mutant, display both severe cell-growth and cell-morphology defects. The same defects are observed with a mutant mimicking permanent phosphorylation of UbK whereas they are not detected for a mutant mimicking defective autophosphorylation of UbK. Moreover, we find that UbK phosphorylation promotes its ability to hydrolyze ATP. These observations show that the hydrolysis of ATP by UbK serves not only for its autophosphorylation but also for a distinct purpose essential for the optimal cell growth and cell-morphogenesis of the pneumococcus. We thus propose a model in which the autophosphorylation/dephosphorylation of UbK regulates its cellular function through a negative feedback loop

Crenarchaeal CdvA Forms Double-Helical Filaments Containing DNA and Interacts with ESCRT-III-Like CdvB

International audienceBACKGROUND: The phylum Crenarchaeota lacks the FtsZ cell division hallmark of bacteria and employs instead Cdv proteins. While CdvB and CdvC are homologues of the eukaryotic ESCRT-III and Vps4 proteins, implicated in membrane fission processes during multivesicular body biogenesis, cytokinesis and budding of some enveloped viruses, little is known about the structure and function of CdvA. Here, we report the biochemical and biophysical characterization of the three Cdv proteins from the hyperthermophilic archaeon Metallospherae sedula. METHODOLOGY/PRINCIPAL FINDINGS: Using sucrose density gradient ultracentrifugation and negative staining electron microscopy, we evidenced for the first time that CdvA forms polymers in association with DNA, similar to known bacterial DNA partitioning proteins. We also observed that, in contrast to full-lengh CdvB that was purified as a monodisperse protein, the C-terminally deleted CdvB construct forms filamentous polymers, a phenomenon previously observed with eukaryotic ESCRT-III proteins. Based on size exclusion chromatography data combined with detection by multi-angle laser light scattering analysis, we demonstrated that CdvC assembles, in a nucleotide-independent way, as homopolymers resembling dodecamers and endowed with ATPase activity in vitro. The interactions between these putative cell division partners were further explored. Thus, besides confirming the previous observations that CdvB interacts with both CdvA and CdvC, our data demonstrate that CdvA/CdvB and CdvC/CdvB interactions are not mutually exclusive. CONCLUSIONS/SIGNIFICANCE: Our data reinforce the concept that Cdv proteins are closely related to the eukaryotic ESCRT-III counterparts and suggest that the organization of the ESCRT-III machinery at the Crenarchaeal cell division septum is organized by CdvA an ancient cytoskeleton protein that might help to coordinate genome segregation

Hijacking of the Pleiotropic Cytokine Interferon-γ by the Type III Secretion System of Yersinia pestis

Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague

Caractérisation d une protéine de fonction inconnue, YdiB de Bacillus subtilis, membre d une nouvelle famille d ATPases exclusivement bactériennes

Nous avons étudié une enzyme de Bacillus subtilis, YdiB, de fonction inconnue. Le gène, spécifiquement procaryote et décrit comme essentiel chez plusieurs espèces, fait de YdiB une cible de choix pour une future recherche d antibiotiques. Nous avons construit une souche délétée de ydiB dont la croissance est fortement réduite. Une faible activité ATPase a été mesurée, néanmoins spécifique de YdiB puisque la mutation d un résidu conservé dans son site actif abolit quasiment toute l activité. Différentes techniques ont révélé que YdiB était capable de former des oligomères, et des résultats similaires ont été observés pour YjeE, son homologue chez E. coli. L addition de sels favorise le déplacement de l équilibre vers le monomère, qui est plus actif que les multimères. Les formes dimériques ont été détectées par cross-link in vivo. Enfin, une recherche des partenaires cellulaires de YdiB suggère un rôle de la protéine lors d une réponse à un stress, ou une interaction avec le ribosome.We have studied an enzyme of Bacillus subtilis, YdiB, of unknown function. The gene, specifically prokaryote and essential in several species, makes YdiB a good target for the research of new anti-bacterial drugs. We made a full knock-out of ydiB in B. subtilis, and its growth was strongly reduced. YdiB exhibited a low ATPase activity that was specific to the protein because it was abrogated by a mutation of an invariant residue in the active site. Different techniques revealed that YdiB forms oligomers, and similar results were found with YjeE, the E. coli orthologue. The addition of salt displaced the equilibrium towards the monomer, that is more active than the dimeric/multimeric form of the enzyme. The dimeric form was also detected by in vivo cross-linking experiments. Finally, we tried to find cellular partners of YdiB and preliminary results suggest a possible role in a stress response, or an interaction with the ribosome.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Caractérisation de 2 transporteurs ABC ( ATP-Binding Cassette ) bactériens de fonction inconnue (YheI/YheH de Bacillus subtilis et Rv1747 de Mycobacterium tuberculosis)

L'émergence du phénotype MDR ( multidrug resistance ) des cellules cancéreuses est souvent corrélée à la surexpression de protéines membranaires appartenant à la superfamille ABC ( ATP binding cassette ). Ces protéines couplent l'hydrolyse de l'ATP au transport d'agents chimiothérapeutiques vers l'extérieur des cellules. Chez les bactéries, des transporteurs homologues ont été impliqués dans certains cas de résistance aux antibiotiques. Deux nouveaux transporteurs ABC bacte riens, Rv1747 de Mycobacterium tuberculosis et YheI/YheH de Bacillus subtilis, potentiellement impliqués dans la résistance aux antibiotiques, ont été étudiés ici en réalisant une expression hétérologue chez Escherichia coli et en isolant des vésicules de membrane inversées. Ce système s'est avéré inapproprié pour l'étude du transporteur Rv1747, à cause vraisemblablement des différences entre E. coli et M. tuberculosis dans l'usage des codons. En revanche, nous avons obtenu un degré important de surexpression de YheI/YheH qui nous a permis de caractériser son activité de transport et d'hydrolyse de l'ATP. Nous avons ainsi montré que les deux protéines, YheI et YheH, s'associent pour former un exportateur hétérodimérique capable de transporter de multiples drogues, et que le rôle des deux sous-unités n'est pas identique dans le mécanisme catalytique du transporteur. Enfin, nous avons réussi à purifier le transporteur YheI/YheH avec un rendement élevé et dans un état fonctionnel stable, permettant d'approfondir sa caractérisation biochimique ainsi que d'obtenir des cristaux bidimensionnels pour une étude structurale par microscopie électronique.The multidrug resistance (MDR) phenotype of cancer cells is mostly associated with overexpression of membrane proteins which belong to the large superfamily of ABC ( ATP binding cassette ) transporters. These proteins couple ATP hydrolysis to the efflux of chemotherapeutic drugs. Homologous transporters from bacteria have been involved in antibiotic resistance. Two new bacterial ABC transporters, Rv1747 from Mycobacterium tuberculosis and YheI/YheH from Bacillus subtilis, putatively involved in antibiotic resistance, have been studied here after heterologous expression in Escherichia coli and isolation of inverted membrane vesicles. This system has proved not to be useful for the study of Rv1747, due probably to codon usage differences between E. coli and M. tuberculosis. By contrast, we reached a high expression level of YheI/YheH which allowed the characterization of its transport and ATP hydrolysis activities. We thus showed that these two proteins associate to form a heterodimeric transporter able to efflux several structurally unrelated drugs, and that the two subunits play different roles in the catalytic mechanism. Finally, we managed to purify YheI/YheH in high yield, in a functional and stable state, which allowed us to extend its biochemical characterization and to obtain 2D crystals for structural studies by electron microscopy.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Découverte d'une nouvelle famille de protéine kinases bactériennes (mécanismes de fonctionnement et rôle cellulaire de YdiB, un archétype chez Baccillus subtilis)

Les données de séquençage des génomes ont révélé une nouvelle famille de protéines UPF0079, comprenant des protéines de fonction inconnue qui sont exclusivement et largement présentes chez les bactéries et qui possèdent un motif A de Walker dans leur séquence. La caractérisation biochimique et l'élucidation du rôle physiologique de cette famille contribueront a élargir nos connaissances en biologie fondamentale, et sont également un préalable vers le développement de nouveaux composés antimicrobiens. Notre étude sur YdiB, un archétype de cette famille chez Bacillus subtilis a révélé à la fois l autophosphorylation de YdiB et son activité de protéine kinase. L activité kinase de double spécificité Ser/ Thr et Tyr de YdiB semble nécessiter son oligomérisation et semble être stimulée par des molécules basiques telles que des polyamines naturelles ou la poly-L-lysine. Les 10 résidus les plus conservés chez cette famille ont été étudiés afin de mieux comprendre le mécanisme moléculaire de YdiB. Concernant la caractérisation fonctionnelle de la phosphorylation liée à YdiB, l étude de l opéron ydiA-B-C-D-E de B. subtilis nous a permis de montrer que YdiB et YdiC fonctionnent comme un couple de protéine kinase/phosphatase de deux protéines substrats dont les fonctions seraient liées aux ribosomes, YdiD et YdiE. Une co-localisation partielle entre YdiB et les ribosomes a été observée. En outre, YdiB est capable de phosphoryler des protéines ribosomiques appartennant aux deux sous-unités 50S et 30S, ainsi que deux GTPases impliquées dans la biogénèse des ribosomes, EngA et EngB. Nous avons également démontré que EngA phosphorylée par YdiB est un substrat in vitro de la phosphatase YdiC. Enfin, basé sur le phosphoprotéome de Bacillus subtilis, des peptides mimant des sites de phosphorylation in vivo ont été utilisés. Certains entre eux sont phosphorylés in vitro par YdiB. Deux de ces peptides appartiennent à la superoxyde dismutase, SodA, dont l'activité in vitro et après purification est régulée positivement via la phosphorylation par YdiB. Nous avons ensuite constaté que les cellules de B. subtilis dépourvues du gène ydiB sont plus sensibles aux agents oxidants tels que le paraquat ou la norfloxacine. Nous proposons que, in vivo, YdiB fonctionne comme une protéine kinase impliquée dans l activité et/ou la stabilité des ribosomes dans des conditions physiologiques normales, et YdiB contribuerait à protéger les cellules contre les dommages du stress oxydatif.Genome sequencing data has revealed genes encoding uncharacterized protein family UPF0079 which are exclusively found in bacteria; broadly distributed in this kingdom and possess an ATP-binding motif in their sequences. Biochemical characterization and physiological role elucidation of UPF0079 will undoubtedly increase our fundamental biology knowledge, and also remain a prerequisite towards the development of new antimicrobial compounds. Our investigation on YdiB, an archetype of this family in Bacillus subtilis revealed both autophosphorylating and protein phosphotransferase activities. The dual-specificity Ser/Thr and Tyr kinase activity of YdiB seems to require oligomerization is upregulated by basic molecule activators such as natural polyamines or poly-L-lysine. The 10 most conserved residues were studied to gain insights into molecular mechanism of the kinase YdiB. To characterize the function of phosphorylation events linked to YdiB, starting with the B. subtilis ydiA-B-C-D-E operon we showed that YdiB and YdiC function as cognate protein kinase/phosphatase towards two ribosome-related protein substrates YdiD and YdiE. Some co-localization between YdiB and ribosomes were observed. Furthermore, YdiB is capable of phosphorylating both ribosomal 50S and 30S subunits as well as two ribosome-binding GTPases EngA and EngB. We also demonstrated that phosphorylated EngA by YdiB is an in vitro substrate of the phosphatase YdiC. Finally, based on the phosphoproteome pf Bacillus subtilis, peptides mimicking the in vivo phosphorylation sites were used. Some of them were found to be phosphorylated in vitro by YdiB, including two peptides which belongs to the superoxide dismutase SodA. The activity of purified SodA was then shown to be upregulated via phosphorylation by YdiB. We furthermore found that B. subtilis cells lacking ydiB become more sensitive to oxidative stress-causing agents such as paraquat or norfloxacin. We propose that in vivo, YdiB functions as a protein kinase involved in ribosome function in normal condition; and in protecting cells from oxidative stress damage.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Interaction between Bacillus subtilis YsxC and ribosomes (or rRNAs).

International audienceYsxC is an essential P-loop GTPase, that binds to the 50S ribosomal subunit, and is required for the proper assembly of the ribosome. The aim of this study was to characterize YsxC ribosome interactions. The stoichiometry of YsxC ribosome subunit complex was evaluated. We showed that YsxC binding to the 50S ribosomal subunit is not affected by GTP, but in the presence of GDP the stoichiometry of YsxC-ribosome is decreased. YsxC GTPase activity was stimulated upon 50S ribosomal subunit binding. In addition, it is shown for the first time that YsxC binds both 16S and 23S ribosomal RNAs

Multidrug ABC transporters in bacteria

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