title: A cationic lumen in the Wzx flippase mediates anionic O-antigen subunit translocation in Pseudomonas aeruginosa PAO1.
abstract: Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Through experimentally based and unbiased topological mapping, our group previously observed that Wzx possesses many charged and aromatic amino acid residues within its 12 transmembrane segments (TMS). Herein, site-directed mutagenesis targeting 102 residues was carried out on the TMS and loops of Wzx, followed by assessment of each construct's ability to restore B-band O-Ag production, identifying eight residues important for flippase function. The importance of various charged and aromatic residues was highlighted, predominantly within the TMS of the protein, revealing functional 'hotspots' within the flippase, particularly within TMS2 and TMS8. Construction of a tertiary structure homology model for Wzx indicated that TMS2 and TMS8 line a central cationic lumen. This is the first report to describe a charged flippase lumen for mediating anionic O-unit translocation across the hydrophobic IM.
authors.affiliation: Department of Molecular and Cellular Biology Biophysics Interdepartmental Group, University of Guelph, Guelph, ON N1G 2W1, Canada.
authors.forename: Salim T
authors.initials: ST
authors.lastname: Islam
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fieldhouse
authors.forename: Erin M
authors.initials: EM
authors.lastname: Anderson
authors.forename: Véronique L
authors.initials: VL
authors.lastname: Taylor
authors.forename: Robert A B
authors.initials: RA
authors.lastname: Keates
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ford
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Lam
mesh_terms: D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D019081:O Antigens / Q000378:metabolism*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics
chemical_list: D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D019081:O Antigens; C095976:O antigen, Pseudomonas