Molecular Basis of Enrofloxacin Affinity to a Membrane Channel of E. Coli - When Binding Does Not Imply Translocation

The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, across the main outer membrane channel OmpF of E. coli is investigated. Through high-resolution ion current fluctuation analysis we count single enrofloxacin channel penetrations, showing a binding to OmpF comparable to the affinity-enhanced translocation through substrate specific channels. A single point mutation D113N increases the dissociation rate 30 times, making the interaction comparable to other antibiotics, corresponding to their weaker binding in a non-specific channel. Molecular dynamics simulations elucidate translocation barriers: WT OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large barrier in the centre, inhibiting antibiotic translocation. Removal of the negative charge on 113 removes the central barrier shifting both peripheral binding sites to one central site enabling translocation. Fluorescence steady-state measurements confirm simulations. Our results demonstrate that a single mutation of the porin results in a substantial modification of translocation. This example demonstrates how translocation through a channel depends not only on the strength of the substrate-channel interaction, but on a local affinity site counteracting the conformational entropy change at the smallest constriction.View Large Image | View Hi-Res Image | Download PowerPoint Slide