Expression and Biochemical Characterization of the Periplasmic Domain of Bacterial Outer Membrane Porin TdeA

  • Kim, Seul-Ki (College of Pharmacy and Research Institute for Drug Development, Pusan National University) ;
  • Yum, Soo-Hwan (College of Pharmacy and Research Institute for Drug Development, Pusan National University) ;
  • Jo, Wol-Soon (Donga-A University Medical Science Research Center) ;
  • Lee, Bok-Luel (College of Pharmacy and Research Institute for Drug Development, Pusan National University) ;
  • Jeong, Min-Ho (Donga-A University Medical Science Research Center) ;
  • Ha, Nam-Chul (College of Pharmacy and Research Institute for Drug Development, Pusan National University)
  • Published : 2008.05.31

Abstract

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic $\alpha$-helical barrel domain and a membrane-embedded $\beta$-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membrane-embedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.

Keywords

References

  1. Choi, J. H., J. I. Choi, and S. Y. Lee. 2005. Display of proteins on the surface of Escherichia coli by C-terminal deletion fusion to the Salmonella typhimurium OmpC. J. Microbiol. Biotechnol. 15: 141-146
  2. Crosby, J. A. and S. C. Kachlany. 2007. TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans. Gene 388: 83-92 https://doi.org/10.1016/j.gene.2006.10.004
  3. DeLano, W. 2002. The PyMOL User's Manual. DeLano Scientific, Palo Alto
  4. Dmitriev, B., F. Toukach, and S. Ehlers. 2005. Towards a comprehensive view of the bacterial cell wall. Trends Microbiol. 13: 569-574 https://doi.org/10.1016/j.tim.2005.10.001
  5. Koronakis, V., J. Li, E. Koronakis, and K. Stauffer. 1997. Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two-dimensional crystals. Mol. Microbiol. 23: 617-626 https://doi.org/10.1046/j.1365-2958.1997.d01-1880.x
  6. Koronakis, V., A. Sharff, E. Koronakis, B. Luisi, and C. Hughes. 2000. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405: 914-919 https://doi.org/10.1038/35016007
  7. Leduc, M., D. Joseleau-Petit, and L. I. Rothfield. 1989. Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells. FEMS Microbiol. Lett. 51: 11-14
  8. Lee, K. E., J. S. Bang, C. H. Baek, D. K. Park, W. Hwang, S. H. Choi, and K. S. Kim. 2007. IVET-based identification of virulence factors in Vibrio vulnificus MO6-24/O. J. Microbiol. Biotechnol. 17: 234-243
  9. Marcyjaniak, M., S. G. Odintsov, I. Sabala, and M. Bochtler. 2004. Peptidoglycan amidase MepA is a LAS metallopeptidase. J. Biol. Chem. 279: 43982-43989 https://doi.org/10.1074/jbc.M406735200
  10. Meroueh, S. O., K. Z. Bencze, D. Hesek, M. Lee, J. F. Fisher, T. L. Stemmler, and S. Mobashery. 2006. Three-dimensional structure of the bacterial cell wall peptidoglycan. Proc. Natl. Acad. Sci. USA 103: 4404-4409
  11. Morona, R., P. A. Manning, and P. Reeves. 1983. Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli. J. Bacteriol. 153: 693-699
  12. Park, J. W., B. R. Je, S. Piao, S. Inamura, Y. Fujimoto, K. Fukase, S. Kusumoto, K. Soderhall, N. C. Ha, and B. L. Lee. 2006. A synthetic peptidoglycan fragment as a competitive inhibitor of the melanization cascade. J. Biol. Chem. 281: 7747-7755 https://doi.org/10.1074/jbc.M510058200
  13. Paulsen, I. T., J. H. Park, P. S. Choi, and M. H. Saier Jr. 1997. A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals from Gram-negative bacteria. FEMS Microbiol. Lett. 156: 1-8 https://doi.org/10.1016/S0378-1097(97)00379-0
  14. Thanabalu, T., E. Koronakis, C. Hughes, and V. Koronakis. 1998. Substrate-induced assembly of a contiguous channel for protein export from E. coli: Reversible bridging of an innermembrane translocase to an outer membrane exit pore. EMBO J. 17: 6487-6496 https://doi.org/10.1093/emboj/17.22.6487
  15. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. CLUSTALX Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882 https://doi.org/10.1093/nar/25.24.4876