Etude structurale et fonctionnelle de la protéine HasB, impliquée dans l'acquisition de l'hème chez Serratia marcescens

Chez les bactéries à Gram négatif, certaines molécules comme les sidérophores ou l hème doivent être activement transportées à travers la membrane externe via des récepteurs membranaires qui permettent la fixation et l internalisation du ligand. Ce type de transport nécessite la protéine de membrane interne TonB qui interagit avec ces récepteurs via un domaine globulaire appelé domaine périplasmique. En plus de la protéine TonB, la bactérie Serratia marcescens possède une protéine homologue, appelée HasB, impliquée dans le système d acquisition de l hème via les hémophores. Ce système est basé sur la sécrétion d une protéine extracellulaire qui capture l hème libre ou lié à des hémoprotéines et qui le délivre ensuite à un récepteur spécifique de la membrane externe (HasR). Chez S. marcescens, les deux protéines HasB et TonB sont actives pour le transport de l hème via le récepteur HasR. Cependant, HasB ne peut pas remplacer TonB pour les autres fonctions TonB-dépendantes. La protéine HasB se comporte donc comme une protéine TonB spécifique de l acquisition de l hème via le récepteur HasR. Afin de comprendre les bases de cette spécificité, l étude structurale par RMN du domaine périplasmique de HasB a été entreprise. Pour cela, les étapes de clonage, d expression et de purification d un échantillon isotopiquement marqué ont été mises au point. L analyse des spectres 3D hétéronucléaires correspondants a permis l attribution des signaux de la protéine ainsi que l identification des éléments de structures secondaires. L interaction entre le domaine périplasmique de HasB et HasR a aussi été étudiée par ITC afin de mieux comprendre la spécificité de cette interaction.In Gram negative bacteria, some molecules such as siderophores or heme must be actively transported through the outer membrane. This transport requires outer membrane receptors which interact and internalize the ligand through the membrane. This transport requires the inner membrane protein TonB, which interacts with these receptors through a globular domain named periplasmic domain. In addition to the TonB protein, the Gram negative bacteria Serratia marcescens possesses a supplementary TonB-like protein called HasB. This protein is a component of the hemophore-dependent heme acquisition system of S. marcescens. This system involves a soluble extracellular protein that acquires free or hemoprotein-bound heme and delivers it to a specific outer membrane receptor (HasR). In S. marcescens both HasB and TonB are active for heme uptake via the HasR receptor. However, HasB can not replace TonB for the others TonB-dependent processes. There is a great deal of evidence that HasB behaves as a specific TonB-like protein dedicated to heme uptake through the HasR receptor. In order to understand the basis of this specificity, NMR structural study of the periplasmic domain of HasB was carried out. Cloning, expression and purification steps of an isotopically labeled sample were optimized. Heteronuclear 3D spectra analysis enabled us to assign backbone and side chain resonances and to localize secondary structure elements. Additionally, the HasB-HasR interaction was studied by ITC microcalorimetry in order to characterize the specificity of this interaction.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Etude structurale et fonctionnelle de la protéine HasS, un facteur anti-sigma impliqué dans la régulation de l'acquisition de l'hème chez Serratia marcescens

Chez les bactéries Gram-négatif, les systèmes d acquisition des différentes sources de fer sont généralement soumis à une régulation très fine. Ils sont régulés en fonction de la concentration intracellulaire en fer. Pour certains, il existe un niveau de régulation supplémentaire qui s effectue par une signalisation transmembranaire. Cette signalisation nécessite l interactions entre trois protéines spécifiques d un système donné : le récepteur membranaire et le facteur sigma de type ECF (extracytoplasmic fonction) et son anti-sigma qui une protéine de membrane interne. Les mécanismes moléculaires de ce processus de signalisation sont inconnus. Durant cette étude, nous nous sommes intéressés à la voie de signalisation régulant l acquisition de l hème via le système Has (heme acquisistion system) de Serratia marcescens. Nous nous sommes focalisés sur la première étape de cette signalisation transmembranaire à savoir, l interaction entre le domaine périplasmique du récepteur HasR et le facteur anti-sigma HasS. Nous avons étudié les aspects structuraux et fonctionnels de ces deux protéines. Nous avons déterminé par RMN la structure 3D du domaine périplasmique de HasR et identifié les résidus impliqués dans la voie de signalisation. De plus, nous avons produit pour la première fois le domaine périplasmique de HasS. Nous avons ensuite déterminé son état de repliement et étudié son interaction avec le domaine périplasmique de HasRIron uptake systems in gram-negative bacteria are generally tightly regulated by iron intracellular concentration. Some of them are also controlled by a transmembrane signaling. Three specific proteins are involved in the latter process : the outer membrane receptor and the ECF (extracytoplasmic function) sigma and antisigma factors. The data about these proteins and of their molecular interactions are sparse and the mechanisms governing this transmembrane signalisation are not understood. We present here the results of the study of the transmembrane signaling in the Has system (heme acquisition system) of Serratia marcescens. We focused on the interaction between the periplasmic domain of the receptor HasR and the ECF anti-sigma factor HasS, two proteins controlling the first step of this signaling process. We carried out structural and functional studies of these protiens. We solved the structure of the periplasmic domain of HasR by NMR and determined which of its residues were involved in the transmembrane signaling. We produced, for the fisrt time, the periplasmic domain of HasS and carried out its characterized regarding its structural features and its interaction with the periplasmic domain of HasRPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB.

International audienceTonB is a cytoplasmic membrane protein required for active transport of various essential substrates such as heme and iron siderophores through the outer membrane receptors of Gram-negative bacteria. This protein spans the periplasm, contacts outer membrane transporters by its C-terminal domain, and transduces energy from the protonmotive force to the transporters. The TonB box, a relatively conserved sequence localized on the periplasmic side of the transporters, has been shown to directly contact TonB. While Serratia marcescens TonB functions with various transporters, HasB, a TonB-like protein, is dedicated to the HasR transporter. HasR acquires heme either freely or via an extracellular heme carrier, the hemophore HasA, that binds to HasR and delivers heme to the transporter. Here, we study the interaction of HasR with a HasB C-terminal domain and compare it with that obtained with a TonB C-terminal fragment. Analysis of the thermodynamic parameters reveals that the interaction mode of HasR with HasB differs from that with TonB, the difference explaining the functional specificity of HasB for HasR. We also demonstrate that the presence of the substrate on the extracellular face of the transporter modifies, via enthalpy-entropy compensation, the interaction with HasB on the periplasmic face. The transmitted signal depends on the nature of the substrate. While the presence of heme on the transporter modifies only slightly the nature of interactions involved between HasR and HasB, hemophore binding on the transporter dramatically changes the interactions and seems to locally stabilize some structural motifs. In both cases, the HasR TonB box is the target for those modifications

Mutagenesis and Molecular Modeling Reveal Three Key Extracellular Loops of the Membrane Receptor HasR That Are Involved in Hemophore HasA Binding▿ †

On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro

Structural Analysis of Proteins from Bacterial Secretion Systems and Their Assemblies by NMR Spectroscopy

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Bonds and Bytes: the Odyssey of Structural Biology

Characterizing structural and dynamic properties of proteins and large macromolecular assemblies is crucial to understand the molecular mechanisms underlying biological functions. In the field of Structural Biology, no single method comprehensively reveals the behavior of biological systems across various spatio-temporal scales. Instead, we have a versatile toolkit of techniques, each contributing a piece to the overall puzzle. Integrative Structural Biology combines different techniques to create accurate and precise multi-scale models that expand our understanding of complex biological systems. This review outlines recent advancements in computational and experimental methods in Structural Biology, with special focus on recent Artificial Intelligence techniques, emphasizes integrative approaches that combine different types of data for precise spatio-temporal modeling, and provides an outlook into future directions of this field

Structure and function of minor pilins of type IV pili

International audienceType IV pili are versatile and highly flexible fibers formed on the surface of many Gram-negative and Gram-positive bacteria. Virulence and infection rate of several pathogenic bacteria, such as Neisseria meningitidis and Pseudomonas aeruginosa, are strongly dependent on the presence of pili as they facilitate the adhesion of the bacteria to the host cell. Disruption of the interactions between the pili and the host cells by targeting proteins involved in this interaction could, therefore, be a treatment strategy. A type IV pilus is primarily composed of multiple copies of protein subunits called major pilins. Additional proteins, called minor pilins, are present in lower abundance, but are essential for the assembly of the pilus or for its specific functions. One class of minor pilins is required to initiate the formation of pili, and may form a complex similar to that identified in the related type II secretion system. Other, species-specific minor pilins in the type IV pilus system have been shown to promote additional functions such as DNA binding, aggregation and adherence. Here, we will review the structure and the function of the minor pilins from type IV pili