Pseudomonas aeruginosa is a Gram-negative bacterium with an intrinsic resistance towards antibiotics due to the lack of a large diffusion pores. Exchange of substances with the environment is done mainly through a set of narrow and substrate-specific porins in its outer membrane that filter molecules according to their size and chemical composition. Among these proteins are OprP and OprO involved in the selective uptake of mono- and pyrophosphates, respectively. Both proteins are homotrimers and each monomer features an hourglass-shaped channel structure including a periplasmic cavity with a lysine cluster. In this study, we focus on the characterization of this lysine cluster in OprO. The importance of these lysine residues was shown with alanine substitutions in single channel conductance experiments, by titration of mono- and pyrophosphate in multi-channel analysis and by molecular dynamics simulations. All obtained data demonstrated that the closer the mutated lysine residues are to arginine 133, the lower gets the single channel conductance. It was found that the ion flow through each monomer can follow two different lysine paths indicating that phosphate ions have a larger freedom on the periplasmic side of the constriction region. Our results emphasize the important role of the lysine residue 121 in the binding site together with arginine 133 and aspartic acid 94. An improved understanding of the ion mobility across these channels can potentially lead to an optimized permeation of (phosphonic acid containing) antibiotics through the outer membrane of P. aeruginosa and the development of new drug molecules.
Keywords: Anion translocation; Black lipid bilayer; Lysine cluster; Molecular dynamics; OprO; Pseudomonas aeruginosa.
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