Topological investigations of the FomA porin from Fusobacterium nucleatum and identification of the constriction loop L6.
Porin FomA in the outer membrane of Fusobacterium nucleatum is a trimeric protein, which exhibits permeability properties similar to that of the well-known enterobacterial diffusion porins. The proposed topology model of the FomA monomer depicts the beta-barrel motif typical of diffusion porins, consisting of 16 antiparallel beta-strands. To investigate the accuracy of the FomA model and assess the topological relationship with other porins, individual deletions of variable size in seven of the eight surface-exposed regions of the porin were genetically engineered. Deletions in the predicted loops L1 to L7 were tolerated by the FomA porins, as judged by a normal assembly in the outer membrane of Escherichia coli and a sustained pore-forming ability. Deletions in the largest proposed external region, loop L6, made the FomA porins considerably more permeable to antibiotics, indicating larger pore channels. The distinctly increased uptake rates and size exclusion limits displayed by the L6 deletion mutant porins, suggest that loop L6 folds back into the beta-barrel thereby constricting the native FomA channel. Thus, the position of the channel constriction loop appears to be shifted towards the C terminus in the FomA porin, as compared to the crystal structures of five non-specific diffusion porins.
Kleivdal
Hans
H
Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1.
Puntervoll
Pål
P
Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1.
Jensen
Harald B
HB
Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1.
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D016967:Fusobacterium nucleatum / Q000737:chemistry* / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D018272:Porins; C083760:fomA protein, Fusobacterium