title: The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel.
abstract: OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000493:pharmacokinetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D015640:Ion Channel Gating; D008081:Liposomes; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D008081:Liposomes; D026901:Membrane Transport Proteins; C402263:OprM protein, Pseudomonas aeruginosa; D014443:Tyrosine