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. 1991 Jun 1;88(11):4877–4881. doi: 10.1073/pnas.88.11.4877

Epitope mapping by mutagenesis distinguishes between the two tertiary structures of the histidine-containing protein HPr.

S Sharma 1, F Georges 1, L T Delbaere 1, J S Lee 1, R E Klevit 1, E B Waygood 1
PMCID: PMC51770  PMID: 1711212

Abstract

Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two-dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two-dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr.

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Selected References

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

  1. Amit A. G., Mariuzza R. A., Phillips S. E., Poljak R. J. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science. 1986 Aug 15;233(4765):747–753. doi: 10.1126/science.2426778. [DOI] [PubMed] [Google Scholar]
  2. Anderson B., Weigel N., Kundig W., Roseman S. Sugar transport. 3. Purification and properties of a phosphocarrier protein (HPr) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli. J Biol Chem. 1971 Nov 25;246(22):7023–7033. [PubMed] [Google Scholar]
  3. Colman P. M., Laver W. G., Varghese J. N., Baker A. T., Tulloch P. A., Air G. M., Webster R. G. Three-dimensional structure of a complex of antibody with influenza virus neuraminidase. 1987 Mar 26-Apr 1Nature. 326(6111):358–363. doi: 10.1038/326358a0. [DOI] [PubMed] [Google Scholar]
  4. Davies D. R., Padlan E. A., Sheriff S. Antibody-antigen complexes. Annu Rev Biochem. 1990;59:439–473. doi: 10.1146/annurev.bi.59.070190.002255. [DOI] [PubMed] [Google Scholar]
  5. Davies D. R., Sheriff S., Padlan E. A. Antibody-antigen complexes. J Biol Chem. 1988 Aug 5;263(22):10541–10544. [PubMed] [Google Scholar]
  6. Delbaere L. T., Bruse L. M., Waygood E. B. Preliminary x-ray data for the HPr protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. J Mol Biol. 1982 May 5;157(1):161–162. doi: 10.1016/0022-2836(82)90519-8. [DOI] [PubMed] [Google Scholar]
  7. Delbaere L. T., Vandonselaar M., Quail J. W., Waygood E. B., Lee J. S. Crystallization of the complex of a monoclonal Fab fragment with the histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. J Biol Chem. 1989 Nov 5;264(31):18645–18646. [PubMed] [Google Scholar]
  8. KUNDIG W., GHOSH S., ROSEMAN S. PHOSPHATE BOUND TO HISTIDINE IN A PROTEIN AS AN INTERMEDIATE IN A NOVEL PHOSPHO-TRANSFERASE SYSTEM. Proc Natl Acad Sci U S A. 1964 Oct;52:1067–1074. doi: 10.1073/pnas.52.4.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Klevit R. E., Drobny G. P., Waygood E. B. Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 1. Sequential resonance assignments. Biochemistry. 1986 Nov 18;25(23):7760–7769. doi: 10.1021/bi00371a071. [DOI] [PubMed] [Google Scholar]
  10. Klevit R. E., Waygood E. B. Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 3. Secondary and tertiary structure as determined by NMR. Biochemistry. 1986 Nov 18;25(23):7774–7781. doi: 10.1021/bi00371a073. [DOI] [PubMed] [Google Scholar]
  11. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Lee L. G., Britton P., Parra F., Boronat A., Kornberg H. Expression of the ptsH+ gene of Escherichia coli cloned on plasmid pBR322. A convenient means for obtaining the histidine-containing carrier protein HPr. FEBS Lett. 1982 Nov 29;149(2):288–292. doi: 10.1016/0014-5793(82)81119-8. [DOI] [PubMed] [Google Scholar]
  14. Lévy S., Zeng G. Q., Danchin A. Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon. Gene. 1990 Jan 31;86(1):27–33. doi: 10.1016/0378-1119(90)90110-d. [DOI] [PubMed] [Google Scholar]
  15. Meadow N. D., Fox D. K., Roseman S. The bacterial phosphoenolpyruvate: glycose phosphotransferase system. Annu Rev Biochem. 1990;59:497–542. doi: 10.1146/annurev.bi.59.070190.002433. [DOI] [PubMed] [Google Scholar]
  16. Postma P. W., Lengeler J. W. Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. Microbiol Rev. 1985 Sep;49(3):232–269. doi: 10.1128/mr.49.3.232-269.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Prasad L., Vandonselaar M., Lee J. S., Delbaere L. T. Structure determination of a monoclonal Fab fragment specific for histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. J Biol Chem. 1988 Feb 15;263(5):2571–2574. [PubMed] [Google Scholar]
  18. Reizer J., Saier M. H., Jr, Deutscher J., Grenier F., Thompson J., Hengstenberg W. The phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: properties, mechanism, and regulation. Crit Rev Microbiol. 1988;15(4):297–338. doi: 10.3109/10408418809104461. [DOI] [PubMed] [Google Scholar]
  19. Roseman S., Meadow N. D. Signal transduction by the bacterial phosphotransferase system. Diauxie and the crr gene (J. Monod revisited). J Biol Chem. 1990 Feb 25;265(6):2993–2996. [PubMed] [Google Scholar]
  20. Sheriff S., Silverton E. W., Padlan E. A., Cohen G. H., Smith-Gill S. J., Finzel B. C., Davies D. R. Three-dimensional structure of an antibody-antigen complex. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8075–8079. doi: 10.1073/pnas.84.22.8075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. WADDELL W. J. A simple ultraviolet spectrophotometric method for the determination of protein. J Lab Clin Med. 1956 Aug;48(2):311–314. [PubMed] [Google Scholar]
  23. Waygood E. B., Erickson E., el Kabbani O. A., Delbaere L. T. Characterization of phosphorylated histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Biochemistry. 1985 Nov 19;24(24):6938–6945. doi: 10.1021/bi00345a028. [DOI] [PubMed] [Google Scholar]
  24. Waygood E. B., Meadow N. D., Roseman S. Modified assay procedures for the phosphotransferase system in enteric bacteria. Anal Biochem. 1979 May;95(1):293–304. doi: 10.1016/0003-2697(79)90219-7. [DOI] [PubMed] [Google Scholar]
  25. Waygood E. B., Reiche B., Hengstenberg W., Lee J. S. Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium. J Bacteriol. 1987 Jun;169(6):2810–2818. doi: 10.1128/jb.169.6.2810-2818.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Waygood E. B., Steeves T. Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. Purification to homogeneity and some properties. Can J Biochem. 1980 Jan;58(1):40–48. doi: 10.1139/o80-006. [DOI] [PubMed] [Google Scholar]
  27. Wright P. E. What can two-dimensional NMR tell us about proteins? Trends Biochem Sci. 1989 Jul;14(7):255–260. doi: 10.1016/0968-0004(89)90058-3. [DOI] [PubMed] [Google Scholar]
  28. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]
  29. el-Kabbani O. A., Waygood E. B., Delbaere L. T. Tertiary structure of histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli. J Biol Chem. 1987 Sep 25;262(27):12926–12929. [PubMed] [Google Scholar]

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