title: Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance.
abstract: The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 2000), demonstrating a distinctive architecture comprising outer membrane beta-barrel and periplasmic helical-barrel structures, which assemble differently from the common beta-barrel-only conformation of porins. Based on their sequence similarity, a similar content of alpha-helical and beta-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the 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 functional domains of OprM and its related family of efflux proteins.
authors: Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
authors: K K
authors: KK
authors: Wong
authors: F S
authors: FS
authors: Brinkman
authors: R S
authors: RS
authors: Benz
authors: R E
authors: RE
authors: Hancock
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D007473:Ion Channels / Q000378:metabolism; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000187:drug effects; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D026901:Membrane Transport Proteins; C402263:OprM protein, Pseudomonas aeruginosa; C073126:tolC protein, E coli