Alteration of pore properties of Escherichia coli OmpF induced by mutation of key residues in anti-loop 3 region

Abstract

The Escherichia coli OmpF pore is governed by an internal constriction consisting of the negatively charged loop 3 folded into the lumen and the positively charged barrel wall located on the opposite side across the pore, 'anti-loop 3'. To investigate the role of anti-loop 3 in solute diffusion, four site-directed mutations, K16A, K16D, R132A and R132D, were introduced into this eyelet region. The mutant porins were expressed efficiently and inserted into the outer membrane, and the thermal stabilities of the resulting trimers were determined. Diffusion of cefepime, a recently developed cephalosporin, was analysed in vivo. In vitro studies were performed on purified porins reconstituted in planar lipid bilayers to measure conductance, selectivity and voltage closure, as well as in liposomes for patch-clamp and sugar-swelling assays. All substitutions modified the ion-channel parameters, and minor conformational changes in the OmpF eyelet region were predicted from modelling studies. Our data show that Lys-16, and to a lesser extent Arg-132, are involved in voltage-gating and pore selectivity via their side-chain charges. Substitution K16D, which causes a severe decrease in critical voltage (V(c)), may generate a channel susceptible to membrane potential, which perturbs cefepime diffusion. These results suggest that the Lys-16 residue plays an important role in the process of diffusion through the OmpF lumen.

Altered pore properties and kinetic changes in mutants of theVibrio choleraePorin OmpU

“…Thus, specific substitutions at the site might have an impact on drug binding, with no other effect on conductance. Our results are in agreement with previous reports that show variability in the correlation between conductance and maltodextrin permeability in OmpF [28,29,36]. Altogether, these results indicate that electrophysiological properties, such as conductance and selectivity, and permeability to specific substrates are governed by specific determinants in the pore's geometry and charge configuration with various degrees of overlap.…”

Physical Insights into Permeation of and Resistance to Antibiotics in Bacteria

“…5, panel III). The fingerprints of this role confirmed the experimental findings of an altered permeability of the pore as a consequence of mutation of the Lys-16 residue [73,74].…”

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

“…Much of the biophysical and mechanistic studies in determining the pathway of antibiotic translocation through porins have focussed on E. coli OmpF, as its structural and functional properties are well understood (Cowan et al, 1992;Danelon et al, 2006). The influx of antibiotics through porins is not just a passive diffusion but involves interactions with key residues in the porin channel and it has been shown in E. coli OmpF that any mutations in these key residues alter the pore properties in terms of diffusion of antibiotics (Bredin et al, 2002;Hajjar et al, 2010b). Hence the crystal structure of porins from different bacterial sources are pre-requisite to understand the specific atomic details, electrostatic pore potential and favourable channel properties involved in antibiotic translocation.…”