title: Determination of the quaternary structure of a bacterial ATP-binding cassette (ABC) transporter in living cells.
abstract: Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that affects patients with cystic fibrosis and immunocompromised individuals. This bacterium coexpresses two unique forms of lipopolysaccharides (LPSs) on its surface, the A- and B-band LPS, which are among the main virulence factors that contribute to its pathogenicity. The polysaccharides in A-band LPSs are synthesized in the cytoplasm and translocated into the periplasm via an ATP-binding cassette (ABC) transporter consisting of a transmembrane protein, Wzm, and a cytoplasmic nucleotide-binding protein, Wzt. Most of the biochemical studies of A-band PSs in Pseudomonas aeruginosa are focused on the stages of the synthesis and ligation of PS, leaving the export stage involving the ABC transporter mostly unexplored. This difficulty is compounded by the fact that the subunit composition and structure of this bi-component ABC transporter are still unknown. Here we propose a simple but powerful method, based on Förster Resonance Energy Transfer (FRET) and optical micro-spectroscopy technology, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of the Wzm-Wzt complex in living cells. It is found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the structure and behavior of this ABC transporter will help develop antibiotics targeting the biosynthesis of the A-band LPS endotoxin.
authors.affiliation: Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA.
authors.forename: Deo R
authors.initials: DR
authors.lastname: Singh
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Mohammad
authors.forename: Suparna
authors.initials: S
authors.lastname: Patowary
authors.forename: Michael R
authors.initials: MR
authors.lastname: Stoneman
authors.forename: Julie A
authors.initials: JA
authors.lastname: Oliver
authors.forename: Liviu
authors.initials: L
authors.lastname: Movileanu
authors.forename: Valerică
authors.initials: V
authors.lastname: Raicu
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000648:ultrastructure*; D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C472370:Wzm protein, Klebsiella pneumoniae; C108433:Wzt protein, bacteria