Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance

The outer membrane protein OprM of Pseudomonas aeruginosa is involved in in trinsic and mutational multiple-antibiotic resistance as part of two resist ance-nodulation-division efflux systems. The crystal structure of TolC, a h omologous protein in Escherichia coli, was recently published (V. Koronakis , A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 200 0), demonstrating a distinctive architecture comprising outer membrane beta -barrel and periplasmic helical-barrel structures, which assemble differen tly from the common beta -barrel-only conformation of porins. Based on thei r sequence similarity, a similar content of alpha -helical and beta -sheet structure determined by circular dichroism spectroscopy, and our observatio n that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model o f OprM was constructed by threading its sequence to the TolC crystal struct ure, Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously i solated insertion mutants of OprM in the context of this model allowed us t o propose roles for different protein domains. Our data indicate that the h elical barrel of the protein is critical for both the function and the inte grity of the protein, while a C-terminal domain localized around the equato rial plane of this helical barrel is dispensable. Extracellular loops appea r to play a lesser role in substrate specificity for this efflux protein co mpared to classical porins, and there appears to be a correlation between t he change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the "iris" mechanism of action for TolC and permit us now to form more focused hypotheses about the funct ional domains of OprM and its related family of efflux proteins.