Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 May 1;363(Pt 3):521–528. doi: 10.1042/0264-6021:3630521

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

Jérôme Bredin 1, Nathalie Saint 1, Monique Malléa 1, Emmanuelle D 1, Gérard Molle 1, Jean-Marie Pagès 1, Valérie Simonet 1
PMCID: PMC1222504  PMID: 11964152

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.

Full Text

The Full Text of this article is available as a PDF (279.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bainbridge G., Mobasheri H., Armstrong G. A., Lea E. J., Lakey J. H. Voltage-gating of Escherichia coli porin: a cystine-scanning mutagenesis study of loop 3. J Mol Biol. 1998 Jan 16;275(2):171–176. doi: 10.1006/jmbi.1997.1474. [DOI] [PubMed] [Google Scholar]
  2. Bauer K., Schmid A., Boos W., Benz R., Tommassen J. Pore formation by pho-controlled outer-membrane proteins of various Enterobacteriaceae in lipid bilayers. Eur J Biochem. 1988 May 16;174(1):199–205. doi: 10.1111/j.1432-1033.1988.tb14082.x. [DOI] [PubMed] [Google Scholar]
  3. Benson S. A., Occi J. L., Sampson B. A. Mutations that alter the pore function of the OmpF porin of Escherichia coli K12. J Mol Biol. 1988 Oct 20;203(4):961–970. doi: 10.1016/0022-2836(88)90121-0. [DOI] [PubMed] [Google Scholar]
  4. Berrier C., Coulombe A., Houssin C., Ghazi A. Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch-clamp. FEBS Lett. 1992 Jul 20;306(2-3):251–256. doi: 10.1016/0014-5793(92)81011-a. [DOI] [PubMed] [Google Scholar]
  5. Chevalier J., Pagès J. M., Malléa M. In vivo modification of porin activity conferring antibiotic resistance to Enterobacter aerogenes. Biochem Biophys Res Commun. 1999 Dec 9;266(1):248–251. doi: 10.1006/bbrc.1999.1795. [DOI] [PubMed] [Google Scholar]
  6. Cowan S. W., Schirmer T., Rummel G., Steiert M., Ghosh R., Pauptit R. A., Jansonius J. N., Rosenbusch J. P. Crystal structures explain functional properties of two E. coli porins. Nature. 1992 Aug 27;358(6389):727–733. doi: 10.1038/358727a0. [DOI] [PubMed] [Google Scholar]
  7. Delcour A. H., Adler J., Kung C. A single amino acid substitution alters conductance and gating of OmpC porin of Escherichia coli. J Membr Biol. 1991 Feb;119(3):267–275. doi: 10.1007/BF01868731. [DOI] [PubMed] [Google Scholar]
  8. Delcour A. H. Function and modulation of bacterial porins: insights from electrophysiology. FEMS Microbiol Lett. 1997 Jun 15;151(2):115–123. doi: 10.1111/j.1574-6968.1997.tb12558.x. [DOI] [PubMed] [Google Scholar]
  9. Delcour A. H., Martinac B., Adler J., Kung C. Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels. Biophys J. 1989 Sep;56(3):631–636. doi: 10.1016/S0006-3495(89)82710-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Delcour A. H., Martinac B., Adler J., Kung C. Voltage-sensitive ion channel of Escherichia coli. J Membr Biol. 1989 Dec;112(3):267–275. doi: 10.1007/BF01870957. [DOI] [PubMed] [Google Scholar]
  11. Fourel D., Bernadac A., Pagès J. M. Involvement of exposed polypeptide loops in trimeric stability and membrane insertion of Escherichia coli OmpF porin. Eur J Biochem. 1994 Jun 1;222(2):625–630. doi: 10.1111/j.1432-1033.1994.tb18905.x. [DOI] [PubMed] [Google Scholar]
  12. Grassi G. G., Grassi C. Cefepime: overview of activity in vitro and in vivo. J Antimicrob Chemother. 1993 Nov;32 (Suppl B):87–94. doi: 10.1093/jac/32.suppl_b.87. [DOI] [PubMed] [Google Scholar]
  13. Guex N., Peitsch M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997 Dec;18(15):2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  14. Im W., Seefeld S., Roux B. A Grand Canonical Monte Carlo-Brownian dynamics algorithm for simulating ion channels. Biophys J. 2000 Aug;79(2):788–801. doi: 10.1016/S0006-3495(00)76336-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iyer R., Wu Z., Woster P. M., Delcour A. H. Molecular basis for the polyamine-ompF porin interactions: inhibitor and mutant studies. J Mol Biol. 2000 Apr 7;297(4):933–945. doi: 10.1006/jmbi.2000.3599. [DOI] [PubMed] [Google Scholar]
  16. Jeanteur D., Schirmer T., Fourel D., Simonet V., Rummel G., Widmer C., Rosenbusch J. P., Pattus F., Pagès J. M. Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10675–10679. doi: 10.1073/pnas.91.22.10675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karshikoff A., Spassov V., Cowan S. W., Ladenstein R., Schirmer T. Electrostatic properties of two porin channels from Escherichia coli. J Mol Biol. 1994 Jul 22;240(4):372–384. doi: 10.1006/jmbi.1994.1451. [DOI] [PubMed] [Google Scholar]
  18. Kleivdal H., Benz R., Tommassen J., Jensen H. B. Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function. Eur J Biochem. 1999 Mar;260(3):818–824. doi: 10.1046/j.1432-1327.1999.00220.x. [DOI] [PubMed] [Google Scholar]
  19. Lakey J. H., Lea E. J., Pattus F. ompC mutants which allow growth on maltodextrins show increased channel size and greater voltage sensitivity. FEBS Lett. 1991 Jan 14;278(1):31–34. doi: 10.1016/0014-5793(91)80076-f. [DOI] [PubMed] [Google Scholar]
  20. Lakey J. H., Pattus F. The voltage-dependent activity of Escherichia coli porins in different planar bilayer reconstitutions. Eur J Biochem. 1989 Dec 8;186(1-2):303–308. doi: 10.1111/j.1432-1033.1989.tb15209.x. [DOI] [PubMed] [Google Scholar]
  21. Lou K. L., Saint N., Prilipov A., Rummel G., Benson S. A., Rosenbusch J. P., Schirmer T. Structural and functional characterization of OmpF porin mutants selected for larger pore size. I. Crystallographic analysis. J Biol Chem. 1996 Aug 23;271(34):20669–20675. [PubMed] [Google Scholar]
  22. Montal M., Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561–3566. doi: 10.1073/pnas.69.12.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Müller D. J., Engel A. Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy. J Mol Biol. 1999 Jan 29;285(4):1347–1351. doi: 10.1006/jmbi.1998.2359. [DOI] [PubMed] [Google Scholar]
  24. Nikaido H., Liu W., Rosenberg E. Y. Outer membrane permeability and beta-lactamase stability of dipolar ionic cephalosporins containing methoxyimino substituents. Antimicrob Agents Chemother. 1990 Feb;34(2):337–342. doi: 10.1128/aac.34.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nikaido H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science. 1994 Apr 15;264(5157):382–388. doi: 10.1126/science.8153625. [DOI] [PubMed] [Google Scholar]
  26. Nikaido H., Rosenberg E. Y. Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J Bacteriol. 1983 Jan;153(1):241–252. doi: 10.1128/jb.153.1.241-252.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pages J. M., Anba J., Bernadac A., Shinagawa H., Nakata A., Lazdunski C. Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coli. Eur J Biochem. 1984 Sep 17;143(3):499–505. doi: 10.1111/j.1432-1033.1984.tb08398.x. [DOI] [PubMed] [Google Scholar]
  28. Phale P. S., Philippsen A., Kiefhaber T., Koebnik R., Phale V. P., Schirmer T., Rosenbusch J. P. Stability of trimeric OmpF porin: the contributions of the latching loop L2. Biochemistry. 1998 Nov 10;37(45):15663–15670. doi: 10.1021/bi981215c. [DOI] [PubMed] [Google Scholar]
  29. Phale P. S., Philippsen A., Widmer C., Phale V. P., Rosenbusch J. P., Schirmer T. Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation. Biochemistry. 2001 May 29;40(21):6319–6325. doi: 10.1021/bi010046k. [DOI] [PubMed] [Google Scholar]
  30. Phale P. S., Schirmer T., Prilipov A., Lou K. L., Hardmeyer A., Rosenbusch J. P. Voltage gating of Escherichia coli porin channels: role of the constriction loop. Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6741–6745. doi: 10.1073/pnas.94.13.6741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prilipov A., Phale P. S., Van Gelder P., Rosenbusch J. P., Koebnik R. Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS Microbiol Lett. 1998 Jun 1;163(1):65–72. doi: 10.1111/j.1574-6968.1998.tb13027.x. [DOI] [PubMed] [Google Scholar]
  32. Przybylski M., Glocker M. O., Nestel U., Schnaible V., Blüggel M., Diederichs K., Weckesser J., Schad M., Schmid A., Welte W. X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance. Protein Sci. 1996 Aug;5(8):1477–1489. doi: 10.1002/pro.5560050804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Saint N., Lou K. L., Widmer C., Luckey M., Schirmer T., Rosenbusch J. P. Structural and functional characterization of OmpF porin mutants selected for larger pore size. II. Functional characterization. J Biol Chem. 1996 Aug 23;271(34):20676–20680. [PubMed] [Google Scholar]
  34. Saint N., Prilipov A., Hardmeyer A., Lou K. L., Schirmer T., Rosenbusch J. P. Replacement of the sole histidinyl residue in OmpF porin from E. coli by threonine (H21T) does not affect channel structure and function. Biochem Biophys Res Commun. 1996 Jun 5;223(1):118–122. doi: 10.1006/bbrc.1996.0855. [DOI] [PubMed] [Google Scholar]
  35. Sali A. Comparative protein modeling by satisfaction of spatial restraints. Mol Med Today. 1995 Sep;1(6):270–277. doi: 10.1016/s1357-4310(95)91170-7. [DOI] [PubMed] [Google Scholar]
  36. Samartzidou H., Delcour A. H. E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF. EMBO J. 1998 Jan 2;17(1):93–100. doi: 10.1093/emboj/17.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saxena K., Drosou V., Maier E., Benz R., Ludwig B. Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin. Biochemistry. 1999 Feb 16;38(7):2206–2212. doi: 10.1021/bi982296f. [DOI] [PubMed] [Google Scholar]
  38. Schindler H., Rosenbusch J. P. Matrix protein from Escherichia coli outer membranes forms voltage-controlled channels in lipid bilayers. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3751–3755. doi: 10.1073/pnas.75.8.3751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schmid B., Maveyraud L., Krömer M., Schulz G. E. Porin mutants with new channel properties. Protein Sci. 1998 Jul;7(7):1603–1611. doi: 10.1002/pro.5560070714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sen K., Hellman J., Nikaido H. Porin channels in intact cells of Escherichia coli are not affected by Donnan potentials across the outer membrane. J Biol Chem. 1988 Jan 25;263(3):1182–1187. [PubMed] [Google Scholar]
  41. Simonet V., Malléa M., Pagès J. M. Substitutions in the eyelet region disrupt cefepime diffusion through the Escherichia coli OmpF channel. Antimicrob Agents Chemother. 2000 Feb;44(2):311–315. doi: 10.1128/aac.44.2.311-315.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Van Gelder P., Saint N., Phale P., Eppens E. F., Prilipov A., van Boxtel R., Rosenbusch J. P., Tommassen J. Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues. J Mol Biol. 1997 Jun 20;269(4):468–472. doi: 10.1006/jmbi.1997.1063. [DOI] [PubMed] [Google Scholar]
  43. Yoshimura F., Nikaido H. Diffusion of beta-lactam antibiotics through the porin channels of Escherichia coli K-12. Antimicrob Agents Chemother. 1985 Jan;27(1):84–92. doi: 10.1128/aac.27.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES