Tracking Membrane Protein Association in Model Membranes

Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue

Analyse structurale et fonctionnelle du domaine ABC "ATP binding cassette" de la protéine HLYB impliquée dans la secretion de l'hemolysine A chez ESCHERICHIA COLI

HlyB, protéine de la membrane interne cytoplasmique â'Escherichia coli, forme un complexe avec HlyD (localisée au niveau de la membrane interne et qui traverse le périplasme) et TolC (protéine de la membrane externe et qui traverse également le périplasme). Ce complexe multiprotéique, traversant les deux membranes d'E. coli avec une stoichiométrie minimale de HlyB, HlyD, TolC, 2:3:3, permet le transport (sécrétion) direct du cytoplasme vers le milieu externe de la toxine hémolysine HlyA. Ce polypeptide (HlyA, 107 kDa) est adressé au complexe de translocation par une séquence signal de sécrétion C-terminale d'environ 46 acides aminés. La sécrétion de HlyA dépend en partie de l'énergie fournie par la force proton motrice et par le transporteur-ABC, HlyB. HlyB, composé d'un domaine transmembranaire et d'un domaine ABC-ATPase cytoplasmique, est également essentielle pour l'oligomérisation de HlyD. Les rôles de HlyD et probablement de TolC, en plus de leur participation intégrale pour la formation d'un canal de transport, ont été mis en évidence à des stades tardifs de sécrétion pour le repliement correct de HlyA avant l'étape finale de son relargage de la surface cellulaire. Pour la translocation de HlyA à travers la membrane cytoplasmique, le domaine de membrane de HlyB est supposé faire partie du canal de transport avec l'énergie d'hydrolyse de l'ATP par HlyB nécessaire au moins pour initier le processus de transport. Afin d'adresser cette question, le domaine ABC-ATPase de HlyB a été purifié, cristallisé et sa structure à haute résolution (2.55 A) a été déterminée. D'autres essais ont également cherché à élucider le rôle du domaine ABC dans la fonction de HlyB par des analyses détaillées in vitro de la régulation de son activité ATPasique . En effet, nous avons établi in vitro d'une part les conditions qui permettent de manière réversible la fixation ou non de l'ATP par le domaine ABC-ATPase et d'autre part les conditions qui contrôlent la transition entre les formes monomère et dimère. Les résultats obtenus, y compris l'analyse des mutants, indiquent que la dimérisation ne serait pas requise à l'activité ATPasique et que in vitro cette activité serait régulée à un niveau de fixation de l'ATP. D'autres études ont également indiqué que le domaine ABC de HlyB purifié interagit de manière spécifique avec la région C-termainale 25 kDa de HlyA via le signal de sécrétion. Un modèle a été proposé pour décrire un nouveau mécanisme de régulation de l'activité ATPasique du transporteur-ABC HlyB en relation avec la fonction de transport de la toxine HlyA.HlyB, an inner, cytoplasmic membrane protein in E. coli, forms a complex with HlyD (anchored in the inner membrane but spanning the periplasm) and TolC (anchored in the outer membrane but also spanning the periplasm). This multiprotein complex, traversing the surface layers of E. coli, with an estimated minimum stoichiometry of HlyB, HlyD, TolC, 2:3:3, provides a transport (secretion) pathway from the cytoplasm to the external medium, for the 107 kDa haemolytic toxin, HlyA. HlyA is targeted to the translocator complex by a specific C-terminal secretion signal sequence of approximately 46 amino-acids. Secretion of HlyA depends upon energy provided by the proton motive force and by the ABC-transporter, HlyB. HlyB, composed of a transmembrane domain and cytoplasmic ABC-ATPase domain, is also essential for the oligomerization of HlyD, which is apparently required for its normal function. At late stages in secretion, HlyD and perhaps TolC are also required for correct folding of HlyA before its release from the cell. For translocation of HlyA across the cytoplasmic membrane, the membrane domain of HlyB is presumed to constitue part of a transport channel, with the energy of hydrolysis of ATP by HlyB required in some way for initiation of the transport process. To address this issue, the NBD (ABC-ATPase) domain of HlyB was purified and crystallized and the crystal structure at 2.55 A is now been determined. Attempts were also made to elucidate the role of the NBD in HlyB function by detailed analysis of the regulation of its ATPase activity in vitro. Thus, conditions were determined which favour the transition of the NBD between monomer and dimer forms and for reversibly switching, on and off, the ATPase activity. The results, including the analysis of mutants, indicate that dimerization is not required for activity and that in vitro, activity is regulated at the level of ATP binding. Other studies also indicate that the purified NBD specifically interacts with the C-terminal 25 kDa of HlyA which contains the C-terminal, secretion signal, essential for recognition of the translocator. A model will be discussed describing a novel mechanism for regulating ATPase activity in this ABC-transporter and its coupling to the transport of HlyA toxin.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Expression, purification, crystallization and preliminary X-ray studies of the outer membrane efflux proteins OprM and OprN from Pseudomonas aeruginosa

Two outer membrane factor family proteins, OprM and OprN, from a tripartite efflux pump found in P. aeruginosa were crystallized. A diffraction data set was collected to 3.8 Å resolution in the space group C2 for OprM crystals

Trimeric structure of OprN and OprM efflux proteins from Pseudomonas aeruginosa, by 2D electron crystallography

OprM and OprN belong to the outer membrane factor family of multidrug efflux proteins from Pseudomonas aeruginosa, a bacterium responsible of nosocomial infections. We report here the two-dimensional (2D) crystallization of OprN and OprM into lipid bilayers and the determination of their 2D projected structure by cryo-electron crystallography, at 1 and 1.4 nm, respectively. Both proteins present a dense ring of protein density, of approximately 7 nm diameter. An additional thin peripheral ring is resolved in OprN structure. Both proteins are assembled as trimers. The results presented here indicate a high structural homology between OprN (and OprM) and TolC, a multidrug efflux protein from Escherichia coli

New OprM structure highlighting the nature of the N-terminal anchor

International audienceAmong the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In Gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved outer membrane factor (OMF) protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications (PTMs), such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional PTM could open new research lines in the field of antibiotics resistance. In Escherichia coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any modification. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X-ray structure at 3.8 Å resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitoylated cysteine

Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis

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