Etude biochimique et structurale du complexe b6f, du complexe membranaire de translocation de la voie Sec et du complexe FhuA-pb5.

A mon arrivée en septembre 1993 au laboratoire de Photosynthèse de l’Institut de Biologie Physico-Chimique dans l’équipe de Jean-Luc Popot, je me suis intéressée à la biochimie des protéines membranaires : i) le mécanisme d’inactivation par les détergents, et ii) la caractérisation en terme de sous-unités (nombre, topologie), cofacteurs, état d’oligomérisation d’un complexe purifié, le b 6 f de la chaîne photosynthétique. Ceci m’a mené à en étudier la structure à partir de cristaux bidimensionnels lors d’un stage post-doctoral à Francfort dans le labo de Werner Kühlbrandt, mais également, à mon retour en France, à développer de nouveaux tensioactifs (hémi)fluorés pour la biochimie des protéines membranaires. De mon séjour en Allemagne j’ai rapporté le goût et la culture de la structure des protéines membranaires, avec la résolution de la structure à 8Å du complexe SecYEG, le translocon de la membrane interne de E. coli. Je m’intéresse donc maintenant également au mécanisme de reconnaissance phage-bactérie et de l’injection de l’ADN phagique dans la bactérie d’un point de vue structural

New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its Escherichia coli receptor FhuA

International audienceThe majority of bacterial viruses are bacteriophages bearing a tail that serves to recognise the bacterial surface and deliver the genome into the host cell. Infection is initiated by the irreversible interaction between the viral receptor binding protein (RBP) and a receptor at the surface of the bacterium. This interaction results ultimately in the phage DNA release in the host cytoplasm. Phage T5 infects Escher-ichia coli after binding of its RBP pb5 to the outer membrane ferrichrome transporter FhuA. Here, we have studied the complex formed by pb5 and FhuA by a variety of biophysical and biochemical techniques. We show that unlike RBPs of known structures, pb5 probably folds as a unique domain fulfilling both functions of binding to the host receptor and interaction with the rest of the phage. Pb5 likely binds to the domain occluding the b-barrel of FhuA as well as to external loops of the barrel. Furthermore, upon binding to FhuA, pb5 undergoes conformational changes, at the secondary and tertiary structure level that would be the key to the transmission of the signal through the tail to the capsid, triggering DNA release. This is the first structural information regarding the binding of a RBP to a proteic receptor. Ó 2012 Elsevier Masson SAS. All rights reserved

Prevalencia de rinotraqueitis infecciosa bovina (IBR) en hatos ganaderos de la parroquia General Proaño, cantón Morona en la provincia de Morona Santiago.

El presente proyecto de investigación tuvo como objetivo determinar la prevalencia de Rinotraqueitis infecciosa bovina (IBR) en hatos ganaderos de la parroquia General Proaño, cantón Morona en la provincia de Morona Santiago. A través de una investigación descriptiva se identificaron las características para identificar la presencia de la enfermedad en los animales con una investigación de campo en los predios de los ganaderos de la parroquia General Proaño; se aplicó un enfoque cuali-cuantitativo para la recolección, procesamiento y análisis de datos. Se utilizó la técnica de encuesta dirigida a los propietarios de los bovinos y la técnica de laboratorio, ELISA de bloqueo, a una población representativa de 158 animales. La prueba de laboratorio ELISA por bloqueo se basa en el bloqueo de la unión de un antisuero mediante anticuerpos presentes en la muestra de sangre tomada del animal infectado. Se determinó que la prevalencia de IBR en bovinos de la parroquia General Proaño es del 23.4% en relación con la población de estudio, Así también, se estratificó la presencia de la enfermedad por sectores de la parroquia, encontrando que el 24.5% de los bovinos de Domono presentaron la enfermedad, General Proaño, cabecera parroquial, registró el 12.2% de casos infectados, Jimbitono indicó un 21.1% y 55.6% en la comunidad de Huacho; también se identificaron casos sospechosos, con un 1.9% en Domono y 2% en Jumbitono. Se concluyó que la prevalencia de la enfermedad se debe al manejo inadecuado de la sanidad en los procesos de vacunación y reproducción de los animales, así como, la alta trashumancia de animales sin certificados sanitarios. Se recomienda a los propietarios y autoridades incrementar las campañas de vacunación y control de la enfermedad a fin de prevenir problemas de salud en los animales.The aim of this research project was to determine the prevalence of Infectious Bovine Rhinotracheitis (IBR) in cattle herds of General Proaño parish, Morona canton in the province of Morona Santiago. Through a descriptive research, the characteristics to identify the presence of the disease in the animals were identified with a field investigation in the cattle ranches of General Proaño parish; a qualitative-quantitative approach was applied for data collection, processing and analysis. The survey technique directed to cattle owners and the laboratory technique, blocking ELISA, was used on a representative population of 158 animals. The blocking ELISA laboratory test is based on blocking the binding of an antiserum by antibodies present in the blood sample taken from the infected animal. It was determined that the prevalence of IBR in cattle in General Proaño parish is 23.4% in relation to the study population. Furthermore, the presence of the disease was stratified by sectors of the parish, finding that 24.5% of the cattle in Domono were infected with IBR. General Proaño, the parish head, registered 12.2% of infected cases, Jimbitono had 21.1% and 55.6% in the community of Huacho; suspected cases were also identified, with 1.9% in Domono and 2% in Jumbitono. It was concluded that the prevalence of the disease is due to inadequate sanitary management in the vaccination and reproduction of animals, as well as the high transhumance of animals without health certificates. It is recommended that owners and authorities increase vaccination and disease control campaigns to prevent animal health problems

Assessing the conformational changes of pb5, the receptor-binding protein of phage T5, upon binding to its Escherichia coli receptor FhuA

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available

The dynamic action of SecA during the initiation of protein translocation

Biotechnology and Biological Sciences Research Council (BBSRC) [a doctoral training grant Ph.D. studentship to S.W. and project grant number BB/I008675/1] and the Wellcome Trust [project grant number 084452]

Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex

The bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA–SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation

The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE-MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE-MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation9FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP11/52067-6; 2017/12436-9; 2013/02451-0FRISBI [ANR-10-INSB-05-02]; GRAL within the Grenoble Partnership for Structural Biology (PSB) [ANR-10-LABX-49-01]; Rhone-Alpes RegionRegion Auvergne-Rhone-Alpes; Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale; fonds FEDER; Centre National de la Recherche Scientifique (CNRS)Centre National de la Recherche Scientifique (CNRS); Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA)French Atomic Energy Commission; University of Grenoble Alpes; EMBL; GIS-Infrastructures en Biologie Sante et Agronomie (IBISA); Laboratoire International Associe BACWALL (CNRS); FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [11/52067-6, 2017/12436-9]; Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-13-BSV8-0015-01]; ANRFrench National Research Agency (ANR); Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale [FDT20160435484]; FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/02451-0

Biogenesis of cytochrome b6 in photosynthetic membranes

In chloroplasts, binding of a c′-heme to cytochrome b6 on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c6 on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b6 in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b6f complexes even in the absence of c′-heme binding to cytochrome b6. Finally, we present a sequential model for apo- to holo-cytochrome b6 maturation integrated within the assembly pathway of b6f complexes in the thylakoid membranes

Plastid thylakoid architecture optimizes photosynthesis in diatoms

Photosynthesis is a unique process that allows independent colonization of the land by plants and of the oceans by phytoplankton. Although the photosynthesis process is well understood in plants, we are still unlocking the mechanisms evolved by phytoplankton to achieve extremely efficient photosynthesis. Here, we combine biochemical, structural and in vivo physiological studies to unravel the structure of the plastid in diatoms, prominent marine eukaryotes. Biochemical and immunolocalization analyses reveal segregation of photosynthetic complexes in the loosely stacked thylakoid membranes typical of diatoms. Separation of photosystems within subdomains minimizes their physical contacts, as required for improved light utilization. Chloroplast 3D reconstruction and in vivo spectroscopy show that these subdomains are interconnected, ensuring fast equilibration of electron carriers for efficient optimum photosynthesis. Thus, diatoms and plants have converged towards a similar functional distribution of the photosystems although via different thylakoid architectures, which likely evolved independently in the land and the ocean.ISSN:2041-172