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A cationic lumen in the Wzx flippase mediates anionic O-antigen subunit translocation in Pseudomonas aeruginosa PAO1.
Mol Microbiol. 2012 Jun; 84(6):1165-76
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15 May 2012
New Finding
DOI: 10.3410/f.715697812.791102865
Studies of the structure/function of Wzx are an important area for understanding lipopolysaccharide (LPS) assembly. The data from this study provide the first evidence that the mechanism of O-antigen being flipped from the inner to the outer leaflet of the inner membrane may involve a lumen/channel...Continue reading
Studies of the structure/function of Wzx are an important area for understanding lipopolysaccharide (LPS) assembly. The data from this study provide the first evidence that the mechanism of O-antigen being flipped from the inner to the outer leaflet of the inner membrane may involve a lumen/channel lined with cationic amino acid residues. Wzx possesses many charged residues within its 12 transmembrane segments. Site-directed mutagenesis of 102 residues followed by activity assessment of each construct allowed the authors to identify 8 residues relevant for the flippase activity.
Disclosures
None declared
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Ramos J: F1000Prime Recommendation of [Islam ST et al., Mol Microbiol 2012, 84(6):1165-76]. In F1000Prime, 15 May 2012; DOI: 10.3410/f.715697812.791102865. F1000Prime.com/715697812#eval791102865
F1000Prime Recommendations, Dissents and Comments for [Islam ST et al., Mol Microbiol 2012, 84(6):1165-76]. In F1000Prime, 18 May 2013; F1000Prime.com/715697812
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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 unit) 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 8 residues important for flippase function. The importance of various charged and aromatic residues were 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. © 2012 Blackwell Publishing Ltd.
© 2012 Blackwell Publishing Ltd.
DOI: 10.1111/j.1365-2958.2012.08084.x
PMID: 22554073
