We characterize the rate-limiting interaction of the antibiotic 10 enrofloxacin with OmpF, a channel from the outer cell wall of Escherichia coli. 11 Reconstitution of a single OmpF trimer into planar lipid membranes allows 12 measurement of the ion current through the channel. Penetration of antibiotics causes 13 ion current blockages, and their frequency allows a conclusion on the kinetics of 14 channel entry and exit. In contrast to other antibiotics, enrofloxacin is able to block the 15 OmpF channel for several milliseconds, reflecting high affinities comparable to 16 substrate-specific channels such as the maltodextrin-specific maltoporin. Surprisingly, 17 the presence of a divalent ion such as Mg2+ leads to fast flickering with an increase in 18 the rates of association and dissociation. All-atom computer modeling provides the 19 most probable pathway able to identify the relevant rate-limiting interaction during antibiotic permeation. Mg2+ has a high affinity 20 for the aspartic acid at the 113 position (D113) in the center of the OmpF intracellular binding site. Therefore, the presence of Mg2+ 21 reverses the charge and enrofloxacin may cross the constriction region in its favorable orientation with the carboxylic group first.

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

CECCARELLI, MATTEO;
2012

Abstract

We characterize the rate-limiting interaction of the antibiotic 10 enrofloxacin with OmpF, a channel from the outer cell wall of Escherichia coli. 11 Reconstitution of a single OmpF trimer into planar lipid membranes allows 12 measurement of the ion current through the channel. Penetration of antibiotics causes 13 ion current blockages, and their frequency allows a conclusion on the kinetics of 14 channel entry and exit. In contrast to other antibiotics, enrofloxacin is able to block the 15 OmpF channel for several milliseconds, reflecting high affinities comparable to 16 substrate-specific channels such as the maltodextrin-specific maltoporin. Surprisingly, 17 the presence of a divalent ion such as Mg2+ leads to fast flickering with an increase in 18 the rates of association and dissociation. All-atom computer modeling provides the 19 most probable pathway able to identify the relevant rate-limiting interaction during antibiotic permeation. Mg2+ has a high affinity 20 for the aspartic acid at the 113 position (D113) in the center of the OmpF intracellular binding site. Therefore, the presence of Mg2+ 21 reverses the charge and enrofloxacin may cross the constriction region in its favorable orientation with the carboxylic group first.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11584/34785
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