The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa.
Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem across the outer membrane. In this study, we examined the effects of deletions in six of the proposed eight surface loops of OprD on the in vivo and in vitro functions of this protein. Native OprD formed very small channels in planar lipid bilayers, with an average single-channel conductance in 1.0 M KCl of 20 pS. When large numbers of OprD channels were incorporated into lipid bilayer membranes, addition of increasing concentrations of imipenem to the bathing solutions resulted in a progressive blocking of the membrane conductance of KCl, indicating the presence of a specific binding site(s) for imipenem in the OprD channel. From these experiments, the concentration of imipenem value of resulting in 50% inhibition of the initial conductance was calculated as approximately 0.6 microM. In contrast, no decrease in channel conductance was observed for the OprDdeltaL2 channel upon addition of up to 2.4 microM imipenem, confirming that external loop 2 was involved in imipenem binding. Deletion of four to eight amino acids from loops 1 and 6 had no effect on antibiotic susceptibility, whereas deletion of eight amino acids from loops 5, 7, and 8 resulted in supersusceptibility to beta-lactams, quinolones, chloramphenicol, and tetracycline. Planar lipid bilayer analysis indicated that the OprDdeltaL5 channel had a 33-fold increase in single-channel conductance in 1 M KCl but had retained its imipenem binding site. The disposition of these loop regions in the interior of the OprD channel is discussed.
Huang
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Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Hancock
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D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D015378:Imipenem / Q000493:pharmacokinetics / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D018272:Porins; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000187:drug effects; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013845:Thienamycins / Q000494:pharmacology*
D001425:Bacterial Outer Membrane Proteins; D018272:Porins; D013845:Thienamycins; C077157:OprD protein, Pseudomonas aeruginosa; D015378:Imipenem