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.

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

“…S7 in the supplemental material). Interestingly, Wzx has a cationic lumen that mediates the translocation of its anionic substrate, the O antigen (62). Our functional data show the essentiality of some positively charged residues located in the MurJ cavity, but it remains to be determined whether the cationic nature of its lumen reflects the ability of MurJ to transport a negatively charged substrate, including lipid II (see Fig.…”

“…Importantly, we demonstrated using SCAM the existence of a central, solventexposed cavity lined by TMDs 1, 2, 7, and 8. This type of architecture is conserved among crystallized MATE proteins (57,58,60) and proposed in a structural model for the O-antigen flippase Wzx (62), despite the low level of primary sequence homology among these proteins. The Wzx proteins belong to the polysaccharide transporter (PST) family of proteins; notably, among the MOP exporter superfamily, MurJ is most related to the PST and MATE families, with the greatest sequence similarity being to PST proteins (31).…”

“…We used two complementary approaches to determine the topology of MurJ experimentally: reporter fusion technology and the substituted cysteine accessibility method (SCAM). Similar approaches have been used for the MOP exporter and O-antigen flippase Wzx (38,62,63). For the reporter strategy, we employed a dual-reporter fusion system (39).…”

Charge Requirements of Lipid II Flippase Activity in Escherichia coli

“…Thus, charged residues that are critical for MurJ function are located strictly in the cavity region. Because the hydrophilic cavity of flippases is likely to serve as the conduit for substrate transport (8,29), we investigated further the requirement for these charged residues.…”

“…These requirements resemble those found in Wzx from Pseudomonas aeruginosa, a MOP exporter superfamily member that flips a trisaccharide linked to undecaprenol-pyrophosphate across the IM. Movement of the anionic trisaccharide moiety through a hydrophilic channel in this flippase is thought to be coordinated by cationic residues in its N-lobe and cavity region (29,41).…”