The Binding of Antibiotics in OmpF Porin

Abstract

The structure of OmpF porin in complex with three common antibiotics (zwitterionic ampicillin, anionic ertapenem, and di-anionic carbenicillin) was determined using X-ray crystallography. The three antibiotics are found to bind within the extracellular and periplasmic pore vestibules, away from the narrow OmpF constriction zone. Using the X-ray structures as a starting point, non-equilibrium MD simulations with an applied membrane voltage show that ionic current through the OmpF channel is blocked with bound ampicillin, but not with bound carbenicillin. The susceptibility of E. coli expressing OmpF mutants to ampicillin and carbenicillin was also experimentally characterized using microbiological assays. These results show that general diffusion by OmpF porins allows for transfer of molecules with varied charged states and give new insights into the design of more efficient antibiotics. A better understanding of this mechanism will shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.

High Salt Concentrations Increase Permeability through OmpC Channels of Escherichia coli

“…The overall properties of single mutants suggest that the ionizable residues exert their influence in a collective way; these results thus appear to imply that the overall configuration of ionizable residues in the channel, and consequently the gross electrostatic property of the channel, possibly plays a decisive role in defining the channel property. The mutation R167L in OmpF was of special interest because Arg 167 was shown to form an "Amp-binding site" together with Arg 168 , Ser 125 , Tyr 32 , and Gly 119 , and the single mutation of R167S was reported to lead to a 20% increase of the susceptibility of the cell to Amp (31). Unfortunately, in our study R167L produced essentially undetectable effects on Amp penetration (Fig.…”

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

“…Similarly we added phosphatidylglycerol lipid (POPG) to the desorption buffer, at a concentration of 5 μ m , and observed up to three lipids bound to OmpF (Figure S3 b). OmpF is also reported to bind to a range of antibiotics within the extracellular and periplasmic pore vestibule 26. Addition of kanamycin (50 μ m ) to the desorption buffer reveals binding of one molecule of the antibiotic per OmpF trimer (Figure S3 c).…”

Extracting Conformational Ensembles of Small Molecules from Molecular Dynamics Simulations: Ampicillin as a Test Case

“…We first assessed the ability of MD simulations: (i) to generate high-diversity conformational ensembles; and (ii) to reproduce the experimentally-available structure of zwitterionic AMP, the one in the co-crystal with OmpF, one of the main general diffusion porins of Escherichia coli [72]. First, we compared the structural clusters representatives of the whole µs-long trajectory with those obtained from a 50 ns-long REMD simulation with 72 replicas in the temperature range 275-600 K. Figure 2 compares the pair-wise root mean square displacement (RMSD) matrix for 30 configurations (all generated vs. all generated) extracted with the two simulations; the picture reports also the corresponding histogram distributions, whose maximum values (mean ± SD) are found to be 5.8 Å (3.2 ± 1.1) and 5.4 Å (3.1 ± 1.0), respectively.…”

“…In order to quantify the ability to generate conformers that are structurally similar to the experimentally-determined structure of AMP [72], we compared the RMSD between the experimental configuration and each of the 30 conformers extracted from our plain MD simulation or generated with the different conformer generator tools considered. The resulting comparison is reported in Figure 4.…”

“…For the four cases considered, the visual comparison between the lowest RMSD structure and the experimental one is reported in the same Figure 4. [72] and each of the 30 conformers extracted from a µs-long MD simulation (red) or generated with FROG2 (blue), OPENBABEL (orange), and RDKIT (green). The boxes, with the same color-codes, show the comparison between the lowest-RMSD structure and the experimental one (blue).…”