Pure dipeptide-binding protein (DppA) from was studied in a filter binding assay to determine its binding specificity. A substrate:DppA stoichiometry of 1:1 was found with both [C]AlaAla and Ala[C]Phe. Surprisingly, substrate binding did not vary over the pH range pH 3–95. Different dipeptides yielded liganded protein with various pI values, implying that DppA can undergo subtly different conformational changes to accommodate different substrates. Using [I]Tyr-peptides as substrates in competition assays, the relative binding affinities for a range of dipeptides were found to parallel their overall transport rates into through the dipeptide permease (Dpp), showing that DppA alone controls the specificity of Dpp. With a series of substituted glycyl peptides, binding affinity was progressively enhanced by alkylation (with methyl to butyl) of the N-terminal α-amino group. Thus, results from this approach provide an essential experimental basis, which complements the information from the crystal structure of DppA, for the design of peptidomimetic antibacterials targeted for transport through Dpp.


Article metrics loading...

Loading full text...

Full text loading...



  1. Abouhamad, W. N. & Manson, M. D. (1994). The dipeptide permease of Escherichia coli closely resembles other bacterial transport systems and shows growth-phase-dependent expression. Mol Microbiol 14, 1077-1092.[CrossRef] [Google Scholar]
  2. Abouhamad, W. N., Manson, M., Gibson, M. M. & Higgins, C. F. (1991). Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium:characterization of the dipeptide permease (Dpp) and the dipeptide-binding protein. Mol Microbiol 5, 1035-1047.[CrossRef] [Google Scholar]
  3. Alves, R. A. & Payne, J. W. (1980). The number and nature of the peptide transport systems of E. coli:characterization of specific transport mutants. Biochem Soc Trans 8, 704-705. [Google Scholar]
  4. Ames, G. F.-L., Mimura, C. S. & Shyamala, V. (1990). Bacterial periplasmic permeases belong to a family of transport proteins operating from E. coli to humans: traffic ATPases. FEMS Microbiol Rev 75, 429-446. [Google Scholar]
  5. Atherton, F. R., Hall, M. J., Hassall, C. H., Lambert, R. W., Lloyd, W. J., Lord, A. V., Ringrose, P. S. & Westmacott, D. (1983). Phosphonopeptides as substrates for peptide transport systems and peptidases of Escherichia coli. Antimicrob Agents Chemother 24, 522-528.[CrossRef] [Google Scholar]
  6. Berger, E. A. & Heppel, L. A. (1972). A binding protein involved in the transport of cystine and diaminopimelic acid in Escherichia coli. J Biol Chem 274, 7684-7694. [Google Scholar]
  7. Blank, V. (1987).Dipeptidchemotaxis in Escherichia coli: Beteiligung des Tap-signaltransducers und des Dpp-transportsystems. Diploma thesis, University of Konstanz, Germany.
  8. Davis, B. D. & Mingioli, E. S. (1950). Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol 60, 17-28. [Google Scholar]
  9. Dunten, P. & Mowbray, S. L. (1995). Crystal structure of dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis. Protein Sci 4, 2327-2334.[CrossRef] [Google Scholar]
  10. Elliot, T. (1993). Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium. J Bacteriol 175, 325-331. [Google Scholar]
  11. Gibson, M. M., Price, M. & Higgins, C. F. (1984). Genetic characterization and molecular cloning of the tripeptide permease (Tpp) genes of Salmonella typhimurium. J Bacteriol 160, 122-130. [Google Scholar]
  12. Goodell, E. W. & Higgins, C. F. (1987). Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli. J Bacteriol 169, 3861-3865. [Google Scholar]
  13. Grimble, G. K. & Backwell, F. R. C. (editors) (1998).Peptides in Mammalian Protein Metabolism: Tissue Utilization and Clinical Targeting. London & Miami: Portland Press.
  14. Guyer, C. A., Morgan, D. G. & Staros, J. V. (1986). Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli. J Bacteriol 168, 775-779. [Google Scholar]
  15. Hammond, S. M., Claesson, A., Jansson, A. M., Larsson, L. G., Pring, B. G., Town, C. M. & Ekstrom, B. (1987). A new class of synthetic antibacterials acting on lipopolysaccharide biosynthesis. Nature 327, 730-732.[CrossRef] [Google Scholar]
  16. Heukeshoven, J. & Dernick, R. (1988). Improved silver staining procedure for fast staining in Phastsystem development unit. Electrophoresis 9, 28-32.[CrossRef] [Google Scholar]
  17. Hiles, I. D., Gallagher, M. P., Jamieson, D. J. & Higgins, C. F. (1987). Molecular characterization of the oligopeptide permease of Salmonella typhimurium. J Mol Biol 195, 125-142.[CrossRef] [Google Scholar]
  18. Igarashi, K., Hiraga, S & Yura, T. (1967). A deoxythymidine kinase deficient mutant of E. coli mapping and transduction studies with phage ϕ80. Genetics 57, 643-654. [Google Scholar]
  19. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef] [Google Scholar]
  20. Manson, M. D., Blank, V., Brade, G. & Higgins, C. F. (1986). Peptide chemotaxis in E. coli involves the Tap signal transducer and the dipeptide permease. Nature 321, 253-256.[CrossRef] [Google Scholar]
  21. Matthews, D. M. & Payne, J. W. (1980). Transmembrane transport of small peptides. Curr Top Membr Transp 14, 331-425. [Google Scholar]
  22. Morley, J. S., Payne, J. W. & Hennessey, T. D. (1983). Antibacterial activity and uptake into Escherichia coli of backbone-modified analogues of small peptides. J Gen Microbiol 129, 3701-3708. [Google Scholar]
  23. Morse, D. E. & Guertin, M. (1972). Amber suA mutations which relieve polarity. J Mol Biol 63, 605-608.[CrossRef] [Google Scholar]
  24. Nakajima, H., Hagting, A., Kunji, E. R. S., Poolman, B. & Konings, W. N. (1997). Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helviticus. Appl Environ Microbiol 63, 2213-2217. [Google Scholar]
  25. Nickitenko, A. V., Trakhanov, S. & Quiocho, F. A. (1995). 2Å resolution structure of DppA, a periplasmic dipeptide transport/chemosensory receptor. Biochemistry 34, 16585-16595.[CrossRef] [Google Scholar]
  26. Olson, E. R., Dunyak, D. S., Jurss, L. M. & Poorman, R. A. (1991). Identification and characterisation of dppA, an E. coli gene encoding a periplasmic dipeptide transport protein. J Bacteriol 173, 234-244. [Google Scholar]
  27. Park, J. T., Debabrata, R., Li, H., Normark, S. & Mengin-Lecreulx, D. (1998). MppA, a periplasmic binding protein essential for import of the bacterial cell wall peptide l-alanyl-γ-d-glutamyl-meso-diaminopimelate. J Bacteriol 180, 1215-1223. [Google Scholar]
  28. Payne, J. W. (1974). Peptide transport in Escherichia coli: permease specificity towards terminal amino group substituents. J Gen Microbiol 80, 269-276.[CrossRef] [Google Scholar]
  29. Payne, J. W. (1980). Transport and utilization of peptides by bacteria. In Microorganisms and Nitrogen Sources, pp. 211-256. Edited by J. W. Payne. Chichester & London: Wiley.
  30. Payne, J. W. (1986). Drug delivery systems: optimising the structure of peptide carriers for synthetic antimicrobial drugs. Drugs Exp Clin Res 12, 585-595. [Google Scholar]
  31. Payne, J. W. (1995). Bacterial peptide permeases as a drug delivery target. In Peptide Based Drug Design: Controlling Transport and Metabolism, pp. 341-367. Edited by M. D. Taylor & G. L. Amidon. Washington, DC: ACS Books.
  32. Payne, J. W. & Bell, G. (1979). Direct determination of the properties of peptide transport systems in Escherichia coli, using a fluorescent-labelling procedure. J Bacteriol 137, 447-455. [Google Scholar]
  33. Payne, J. W. & Gilvarg, C. (1968). Role of the terminal carboxyl group in peptide transport in Escherichia coli. J Biol Chem 243, 335-340. [Google Scholar]
  34. Payne, J. W. & Nisbet, T. M. (1980). Limitations to the use of radioactively-labelled substrates for studying peptide transport in microorganisms. FEBS Lett 119, 73-76.[CrossRef] [Google Scholar]
  35. Payne, J. W. & Smith, M. W. (1994). Peptide transport by microorganisms. Adv Microb Physiol 36, 1-80. [Google Scholar]
  36. Payne, J. W., Morley, J. S., Armitage, P. & Payne, G. M. (1984). Transport and hydrolysis of antibacterial peptide analogues in Escherichia coli: backbone-modified aminoxy peptides. J Gen Microbiol 130, 2253-2265. [Google Scholar]
  37. Perry, D. & Gilvarg, C. (1984). Spectrophotometric determination of affinities of peptides for their transport systems in Escherichia coli. J Bacteriol 160, 943-948. [Google Scholar]
  38. Pugsley, A. P. & Reeves, P. (1976). Iron uptake in colicin B resistant mutants of Escherichia coli K12. J Bacteriol 126, 1052-1062. [Google Scholar]
  39. Richarme, G. & Kepes, A. (1983). Study of binding protein–ligand interaction by ammonium sulphate-assisted adsorption on cellulose ester filters. Biochim Biophys Acta 742, 16-24.[CrossRef] [Google Scholar]
  40. Smith, M. W. (1992).Characterization and exploitation of microbial peptide transport systems. PhD thesis, University of Wales, Bangor.
  41. Smith, M. W. & Payne, J. W. (1990). Simultaneous exploitation of different peptide permeases by combinations of synthetic peptide smugglins can lead to enhanced antibacterial activity. FEMS Microbiol Lett 70, 311-316.[CrossRef] [Google Scholar]
  42. Steiner, H. Y., Naider, F. & Becker, J. M. (1995). The PTR family – a new group of peptide transporters. Mol Microbiol 16, 825-834.[CrossRef] [Google Scholar]
  43. Tame, J. R. H., Murshudov, G. N., Dodson, E. J., Neil, T. K., Dodson, G. G., Higgins, C. F. & Wilkinson, A. J. (1994). The structural basis of sequence-independent peptide binding by OppA protein. Science 264, 1578-1581.[CrossRef] [Google Scholar]
  44. Tame, J. R. H., Dodson, E. J., Marshudov, G., Higgins, C. F. & Wilkinson, A. J. (1995). The crystal structures of the oligopeptide-binding protein OppA complexed with tripeptide and tetrapeptide ligands. Structure 3, 1395-1406.[CrossRef] [Google Scholar]
  45. Taylor, M. D. & Amidon, G. L. (editors) (1995).Peptide-based Drug Design: Controlling Transport and Metabolism. Washington, DC: American Chemical Society.
  46. Tyreman, D. R., Smith, M. W., Payne, G. M. & Payne, J. W. (1992). Exploitation of peptide transport systems in the design of antimicrobial agents. In Molecular Aspects of Chemotherapy, pp. 127–142. Edited by D. Shugar, W. Rode & E. Borowski. Berlin: Springer.
  47. Tyreman, D. R., Smith, M. W., Marshall, N. J., Payne, G. M., Schuster, C. F., Grail, B. M. & Payne, J. W. (1998). Peptides as prodrugs: the smugglin concept. In Peptides in Mammalian Protein Metabolism: Tissue Utilisation and Clinical Targeting, pp. 141-157. Edited by G. K. Grimble & F. R. C. Backwell. London: Portland Press.
  48. Verkamp, E., Bachman, V. M., Bjornsson, J. M., Soll, D. & Eggertsson, G. (1993). The periplasmic dipeptide permease system transports 5-aminolevulinic acid in Escherichia coli. J Bacteriol 175, 1452-1456. [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error