The Transmembrane Domain of a Bicomponent ABC Transporter Exhibits Channel-Forming Activity
journal contributionposted on 2016-07-05, 00:00 authored by Mohammad M. Mohammad, Noriko Tomita, Makoto Ohta, Liviu Movileanu
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.
Wzt polypeptideTransmembrane DomainBicomponent ABC Transporter Exhibits Channel-Forming Activity Pseudomonas aeruginosalatter polypeptideLPSWzm-based proteinWzm proteinABC proteinchannel-like activityA-band polysaccharideschannel-forming abilityATPase activitybicomponent ATP-binding cassettebicomponent ABC transporterTEMNBD Wzt proteinWzt domain proteinstransmission electron microscopyA-band PSslipid membraneTMD Wzm protein200 mM KCl solutiontransmembrane Wzm proteinring-shaped structure