Molecular Model of Norfloxacin Translocation Through the Yersinia pseudotuberculosis OmpF Porin Channel

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Abstract

The interaction of Yersinia pseudotuberculosis OmpF (YpOmpF) porin with the fluoroquinolone antibiotic norfloxacin (Nf) and its derivatives (mono- and dihydrochloride) was studied using methods based on the use of bilayer lipid membranes (BLM), molecular modeling, and antibacterial activity testing. Asymmetric behavior of charged Nf (NfH+1) and (Nf2H+2) molecules was found to move through the YpOmpF channel depending on the membrane voltage and the side of antibiotic addition. Electrophysiological data were confirmed by computer modeling. For charged forms of the antibiotic, the presence of two peripheral high-affinity binding sites (NBS1 and NBS2), as well as an asymmetric current blocking site near the channel constriction zone (NBS3), was detected. The NBS1 site located near the channel mouth has almost the same affinity for both charged forms of Nf, while the localization of the more energetically favorable NBS2 site for the two salt forms of the antibiotic differs significantly. Nf has only one binding site near the channel constriction zone, which is a cluster of sites with lower overall affinity compared to the peripheral binding sites mentioned above. Slight differences were found in the antibacterial activity of the three forms of Nf, which is likely due to their different charge states and, accordingly, different permeability and/or ability to bind within the YpOmpF channel.

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D. K. Chistyulin

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Email: zel01@mail.ru
Russian Federation, Vladivostok, 690022

E. A. Zelepuga

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Author for correspondence.
Email: zel01@mail.ru
Russian Federation, Vladivostok, 690022

V. L. Novikov

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Email: zel01@mail.ru
Russian Federation, Vladivostok, 690022

N. N. Balaneva

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Email: zel01@mail.ru
Russian Federation, Vladivostok, 690022

V. P. Glazunov

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Email: zel01@mail.ru
Russian Federation, Vladivostok, 690022

E. A. Chingizova

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Email: zel01@mail.ru
Russian Federation, Vladivostok, 690022

V. A. Khomenko

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Email: zel01@mail.ru
Russian Federation, Vladivostok, 690022

O. D. Novikova

Еlyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences

Email: novolga_05@mail.ru
Russian Federation, Vladivostok, 690022

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Supplementary files

Supplementary Files
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2. Fig. 1. Structural formulas of norfloxacin (1) and its salts, monohydrochloride (2) and dihydrochloride (3).

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3. Fig. 2. Structures of the four theoretically possible forms of the norfloxacin molecule.

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4. Fig. 3. Total conductance of YpOmpF porin channels in the absence (left part) and upon addition (right part) of norfloxacin monohydrochloride (a). Current recording through a single YpOmpF channel in the presence of antibiotic (b). Aqueous phase: 1 M KCl, 10 mM Tris-HCl, 10 mM MES, 10 mM beta-alanine, 200 ng/mL protein (a) and 20 ng/mL (b). Membrane potential 50 mV (a) and -100 to +100 mV (b). On the ordinate axis is the ionic current through the membrane, mA.

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5. Fig. 4. Results of electrophysiological experiments with norfloxacin monohydrochloride (b and c) and dihydrochloride (d and e) on single channels of the non-specific porin OmpF of Y. pseudotuberculosis.

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6. Fig. 5. Spatial organization of probable complexes of YpOmpF with Nf-HCl. 3D structure of the YpOmpF porin homotrimer, two subunits are represented as a molecular surface, one as a ribbon diagram, lipids and water surroundings have been removed for clarity. NfH+1 (a) and Nf2H+2 (b) molecules in the two binding sites are shown in globular representation, in the trans-position in pink and blue, in the cis-position in yellow and green, respectively, the surrounding amino acid residues are shown in rod representation. 2D-diagrams of intermolecular interactions of NfH+1 (a) and Nf2H+2 (b) in both binding sites are shown in the footnotes.

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7. Fig. 6. Spatial organization of probable complexes of YpOmpF with NfH+1 and Nf2H+2. 3D structure of the porin monomer YpOmpF, as a ribbon diagram, part of the β-strand, lipids and aqueous surroundings have been removed for clarity. NfH+1 (blue, bottom inset) and Nf2H+2 (brown, top inset) molecules in the NBS3 binding site are shown in a spherical rod representation. 2D diagrams of noncovalent intermolecular interactions of NfH+1 (upper inset) and Nf2H+2 (lower inset) with YpOmpF are shown in the footnotes.

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8. Fig. 7. Spatial organization of probable complexes of Nf with YpOmpF. 3D structure of the porin monomer YpOmpF, in the form of a ribbon diagram, part of the β-tethers, lipids and aqueous surroundings have been removed for clarity. The possible orientations of the Nf molecule (top part in pink and bottom part in blue) in the EQ are given in spherical rod representation, residues responsible for Nf binding are given in rod representation and labeled. Ionic interactions are indicated by blue surfaces, hydrogen bonds by gray rods.

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9. Fig. 8. Energy barrier profiles during translocation of charged forms of norfloxacin (NfH+1 and Nf2H+2) through the OmpF channel of Y. pseudotuberculosis.

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