Alteration of pore properties of Escherichia coli OmpF induced by mutation of key residues in anti-loop 3 region.
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.
Bredin
Jérôme
J
CJF9606-EA2197, IFR 48, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France.
Saint
Nathalie
N
Malléa
Monique
M
Dé
Emmanuelle
E
Molle
Gérard
G
Pagès
Jean-Marie
JM
Simonet
Valérie
V
D001120:Arginine / Q000378:metabolism; D000077723:Cefepime; D002511:Cephalosporins / Q000378:metabolism; D004058:Diffusion; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015640:Ion Channel Gating; D008051:Lipid Bilayers; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
D002511:Cephalosporins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; C109237:OmpF protein; D018272:Porins; D000077723:Cefepime; D001120:Arginine; D008239:Lysine