Antibiotic Permeation across the OmpF Channel: Modulation of the Affinity Site in the Presence of Magnesium

Abstract

We characterize the rate-limiting interaction of the antibiotic enrofloxacin with OmpF, a channel from the outer cell wall of Escherichia coli . Reconstitution of a single OmpF trimer into planar lipid membranes allows measurement of the ion current through the channel. Penetration of antibiotics causes ion current blockages, and their frequency allows a conclusion on the kinetics of channel entry and exit. In contrast to other antibiotics, enrofloxacin is able to block the OmpF channel for several milliseconds, reflecting high affinities comparable to substrate-specific channels such as the maltodextrin-specific maltoporin. Surprisingly, the presence of a divalent ion such as Mg(2+) leads to fast flickering with an increase in the rates of association and dissociation. All-atom computer modeling provides the most probable pathway able to identify the relevant rate-limiting interaction during antibiotic permeation. Mg(2+) has a high affinity for the aspartic acid at the 113 position (D113) in the center of the OmpF intracellular binding site. Therefore, the presence of Mg(2+) reverses the charge and enrofloxacin may cross the constriction region in its favorable orientation with the carboxylic group first.

Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli

“…Solvent-free Lipid Bilayer Measurements-Reconstitution experiments and noise analysis were performed as described previously in detail (19). The Montal and Muller technique was used to form phospholipid bilayer using 1,2-diphytanoyl-snglycero-3-phosphocholine (Avanti Polar Lipids) (23).…”

“…The authors suggested that, in contrast to the obvious exclusion due to reduced channel size, effects such as changes in the electric field at the constriction zone might have an impact on the channel permeability (17). Recent computational and experimental studies also suggested that the electrostatic profile inside the channel might play a major role in transit of polar molecules (18,19). Subsequently, the internal electrostatics of the mutant porins (OmpC20 and OmpC33) were analyzed in the absence of any antibiotic or metabolite, using water as a probe to sense the internal electric field (20).…”

Physical methods to quantify small antibiotic molecules uptake into Gram-negative bacteria

“…Modelling revealed that Mg 2+ can bind strongly in the eyelet region of OmpF, as shown in Fig. 3, reversing the net charge of this region [21]. As a consequence, enrofloxacin can penetrate with an optimal orientation for permeation, his carboxylic group entering first, increasing the association rate and the total flux, as measured by electrophysiology.…”

“…In contrast, addition of 5 mM MgCl 2 into the buffer change completely the spectra, many ion current blockages are visible (Fig. 2 bottom) [21]. The example of Fig.…”

How do the protonation states of E296 and D312 in OmpF and D299 and D315 in homologous OmpC affect protein structure and dynamics? Simulation studies

“…It also showed that L3 flexibility affected a change in pore cavity [25]. MD simulations were also successfully used to observe the solute (such as antibiotics) passage through OmpF in comparison with experiments [2, 15,[29][30][31]. OmpF are computationally well studied whereas few computational studies on OmpC are found.…”