title: The Transmembrane Domain of a Bicomponent ABC Transporter Exhibits Channel-Forming Activity.
abstract: Pseudomonas aeruginosa is an opportunistic pathogen that expresses two unique forms of lipopolysaccharides (LPSs) on its bacterial surface, the A- and B-bands. The A-band polysaccharides (A-band PSs) are thought to be exported into the periplasm via a bicomponent ATP-binding cassette (ABC) transporter located within the inner membrane. This ABC protein complex consists of the transmembrane (TMD) Wzm and nucleotide-binding (NBD) Wzt domain proteins. Here, we were able to probe ∼1.36 nS-average conductance openings of the Wzm-based protein complex when reconstituted into a lipid membrane buffered by a 200 mM KCl solution, demonstrating the large-conductance, channel-forming ability of the TMDs. In agreement with this finding, transmission electron microscopy (TEM) imaging revealed the ring-shaped structure of the transmembrane Wzm protein complex. As hypothesized, using liposomes, we demonstrated that Wzm interacts with Wzt. Further, the Wzt polypeptide indeed hydrolyzed ATP but exhibited a ∼75% reduction in the ATPase activity when its Walker A domain was deleted. The distribution and average unitary conductance of the TMD Wzm protein complex were altered by the presence of the NBD Wzt protein, confirming the regulatory role of the latter polypeptide. To our knowledge, the large-conductance, channel-like activity of the Wzm protein complex, although often hypothesized, has not previously been demonstrated. These results constitute a platform for future structural, biophysical, and functional explorations of this bicomponent ABC transporter.
authors.affiliation: Department of Physics, Syracuse University , 201 Physics Building, Syracuse, New York 13244-1130, United States.
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Mohammad
authors.affiliation: Department of Physics, Syracuse University , 201 Physics Building, Syracuse, New York 13244-1130, United States.|Institute of Fluid Science, Tohoku University , 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Tomita
authors.affiliation: Institute of Fluid Science, Tohoku University , 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ohta
authors.affiliation: Department of Physics, Syracuse University , 201 Physics Building, Syracuse, New York 13244-1130, United States.|Structural Biology, Biochemistry, and Biophysics Program, Syracuse University , 111 College Place, Syracuse, New York 13244-4100, United States.|The Syracuse Biomaterials Institute, Syracuse University , 121 Link Hall, Syracuse, New York 13244, United States.
authors.forename: Liviu
authors.initials: L
authors.lastname: Movileanu
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D003545:Cysteine / Q000235:genetics; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D003545:Cysteine