Bridging timescales and length scales: from macroscopic flux to the molecular mechanism of antibiotic diffusion through porins

Abstract

Our aim in this study was to provide an atomic description of ampicillin translocation through OmpF, the major outer membrane channel in Escherichia coli and main entry point for beta-lactam antibiotics. By applying metadynamics simulations, we also obtained the energy barriers along the diffusion pathway. We then studied the effect of mutations that affect the charge and size at the channel constriction zone, and found that in comparison to the wild-type, much lower energy barriers are required for translocation. The expected higher translocation rates were confirmed on the macroscopic scale by liposome-swelling assays. A microscopic view on the millisecond timescale was obtained by analysis of temperature-dependent ion current fluctuations in the presence of ampicillin and provide the enthalpic part of the energy barrier. By studying antibiotic translocation over various timescales and length scales, we were able to discern its molecular mechanism and rate-limiting interactions, and draw biologically relevant conclusions that may help in the design of drugs with enhanced permeation rates.

Figure 1

Structural details of OmpF. (A) The backbone of OmpF is displayed in gray cartoons. The charged residues at the constriction region (D113, E117, and D121 on the L3 side, and R42, R82, and R132 on the anti-L3 side) are colored by residue type (positively charged in blue, negatively charged in red). (B) The OmpF structure is displayed in gray molecular surface to highlight the space available. Loop L3 is colored in orange and the charged residues at the constriction region are colored as in A.

Figure 2

Typical tracks of ion current through a single WT OmpF channel and mutants D113N and R132A, reconstituted into DPhPC lipid membranes in the presence of 10 mM ampicillin and 1 M KCl at pH 6. Applied voltage is 50 mV.

Figure 3

(A) Statistical analysis revealing temperature-dependent blocking events of ampicillin with WT OmpF and D113N. The continuous line represents the exponential fit. (B) Effect of temperature on the antibiotic residence time (τ) for WT OmpF and D113N mutant.

Figure 4

One-dimensional free-energy profiles for the translocation of ampicillin through WT OmpF (A), D113N (B), and R132A (C). The minima at the constriction region are highlighted in gray and the energy barriers are reported in kT. The “exit” label refers to the periplasmic side.

Figure 5

Liposome-swelling assay with proteoliposomes containing WT OmpF or mutants D113N and R132A. Arabinose, a small molecule that is able to penetrate perfectly, was used as a reference to normalize the swelling rates (=100%). A second control was performed with raffinose, which does not permeate through OmpF.

Figure 6

Molecular details (side views) of ampicillin at the binding site of the constriction region of (A) WT OmpF, (B) D113N, and (C) R132A. The views and orientations in this figure are the same as in Fig. 1 (the top is toward the vestibule, the bottom is toward the periplasmic space). The antibiotic is displayed in stick representation and colored by atom type (blue for nitrogen, red for oxygen, cyan for carbon) where hydrogens are not shown. The backbone of OmpF is displayed in gray cartoons to highlight its secondary structures. The constriction region is highlighted by loop L3 (colored in orange). Residues of OmpF that are seen as strongly interacting with the antibiotic are labeled using the one-letter amino acid code; those making H-bonds are colored by residue type (positively charged in blue, negatively charged in red, polar in green), and those making hydrophobic contacts are displayed with their molecular surface, highlighting their shape.

Figure 7

Molecular details (top view) from equilibrium simulations started at Minima-II for WT OmpF (A), D113N (B), and R132A (C) mutants. Ampicillin is displayed in stick representation and colored according to atom type. The backbone of OmpF is shown by gray cartoons (L3 is colored orange). The residues making H-bonds are colored by residue type, and those making hydrophobic contacts are displayed by gray molecular surface. Below, the average SASA is reported for WT OmpF (D), D113N (E), and R132A (F) in the presence (black) and absence (red) of ampicillin.