Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function

FomA porin is the major outer-membrane protein of Fusobacterium nucleatum. It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins. According to the proposed topology model, each monomer of this trimeric protein is a beta-barrel consisting o f 16 transmembrane segments with eight surface-exposed loops. Several conse rved charged residues are proposed to extend from the beta-barrel wall into the aqueous channel lumen, and may contribute to a transverse electric fie ld similar to that at the pore constriction of porins with known structure. The goal of our study was to identify particular basic residues contributi ng to such an electric field in FomA. Several arginines and lysines were re placed by negatively charged glutamates or uncharged alanines. The mutated FomA porins were expressed in Escherichia coli, and the effects on pore fun ction were studied in vivo, by assaying the uptake rate of beta-lactam anti biotics, and in vitro after reconstitution of the purified proteins in lipi d bilayer membranes. Some of the point mutations had a significant impact o n the channel properties. The substitution R92A produced a 130% increased p ermeability of the zwitterionic beta-lactam cephaloridine, and the cation s electivity of R92E increased by 70%. The effects of the R90E substitution o n channel properties were similar. Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity. The la tter mutation had no effect on cation selectivity, but the K78A substitutio n improved the uptake rate of cephaloridine. The results presented here ind icate that arginines 90 and 92 are probably part of the constriction zone o f the FomA porin, and lysine 78 and arginines 115 and 117 are probably in c lose proximity to this region as well.