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. 1993 Apr;2(4):543–558. doi: 10.1002/pro.5560020406

Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

J G Pelton 1, D A Torchia 1, N D Meadow 1, S Roseman 1
PMCID: PMC2142369  PMID: 8518729

Abstract

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.

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Selected References

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  1. Bachovchin W. W. 15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. Biochemistry. 1986 Nov 18;25(23):7751–7759. doi: 10.1021/bi00371a070. [DOI] [PubMed] [Google Scholar]
  2. Beneski D. A., Nakazawa A., Weigel N., Hartman P. E., Roseman S. Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a phosphocarrier protein HPr from wild type and mutants of Salmonella typhimurium. J Biol Chem. 1982 Dec 10;257(23):14492–14498. [PubMed] [Google Scholar]
  3. Blomberg F., Maurer W., Rüterjans H. Nuclear magnetic resonance investigation of 15N-labeled histidine in aqueous solution. J Am Chem Soc. 1977 Dec 7;99(25):8149–8159. doi: 10.1021/ja00467a005. [DOI] [PubMed] [Google Scholar]
  4. Clore G. M., Bax A., Driscoll P. C., Wingfield P. T., Gronenborn A. M. Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy. Biochemistry. 1990 Sep 4;29(35):8172–8184. doi: 10.1021/bi00487a027. [DOI] [PubMed] [Google Scholar]
  5. Clore G. M., Gronenborn A. M. Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy. Science. 1991 Jun 7;252(5011):1390–1399. doi: 10.1126/science.2047852. [DOI] [PubMed] [Google Scholar]
  6. Dao-pin S., Anderson D. E., Baase W. A., Dahlquist F. W., Matthews B. W. Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. Biochemistry. 1991 Dec 10;30(49):11521–11529. doi: 10.1021/bi00113a006. [DOI] [PubMed] [Google Scholar]
  7. Dörschug M., Frank R., Kalbitzer H. R., Hengstenberg W., Deutscher J. Phosphoenolpyruvate-dependent phosphorylation site in enzyme IIIglc of the Escherichia coli phosphotransferase system. Eur J Biochem. 1984 Oct 1;144(1):113–119. doi: 10.1111/j.1432-1033.1984.tb08438.x. [DOI] [PubMed] [Google Scholar]
  8. Fairbrother W. J., Cavanagh J., Dyson H. J., Palmer A. G., 3rd, Sutrina S. L., Reizer J., Saier M. H., Jr, Wright P. E. Polypeptide backbone resonance assignments and secondary structure of Bacillus subtilis enzyme IIIglc determined by two-dimensional and three-dimensional heteronuclear NMR spectroscopy. Biochemistry. 1991 Jul 16;30(28):6896–6907. doi: 10.1021/bi00242a013. [DOI] [PubMed] [Google Scholar]
  9. Fairbrother W. J., Gippert G. P., Reizer J., Saier M. H., Jr, Wright P. E. Low resolution solution structure of the Bacillus subtilis glucose permease IIA domain derived from heteronuclear three-dimensional NMR spectroscopy. FEBS Lett. 1992 Jan 20;296(2):148–152. doi: 10.1016/0014-5793(92)80367-p. [DOI] [PubMed] [Google Scholar]
  10. Fairbrother W. J., Palmer A. G., 3rd, Rance M., Reizer J., Saier M. H., Jr, Wright P. E. Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy. Biochemistry. 1992 May 12;31(18):4413–4425. doi: 10.1021/bi00133a005. [DOI] [PubMed] [Google Scholar]
  11. Fesik S. W., Zuiderweg E. R. Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules. Q Rev Biophys. 1990 May;23(2):97–131. doi: 10.1017/s0033583500005515. [DOI] [PubMed] [Google Scholar]
  12. Hammen P. K., Waygood E. B., Klevit R. E. Reexamination of the secondary and tertiary structure of histidine-containing protein from Escherichia coli by homonuclear and heteronuclear NMR spectroscopy. Biochemistry. 1991 Dec 24;30(51):11842–11850. doi: 10.1021/bi00115a014. [DOI] [PubMed] [Google Scholar]
  13. Herzberg O., Reddy P., Sutrina S., Saier M. H., Jr, Reizer J., Kapadia G. Structure of the histidine-containing phosphocarrier protein HPr from Bacillus subtilis at 2.0-A resolution. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2499–2503. doi: 10.1073/pnas.89.6.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hol W. G. The role of the alpha-helix dipole in protein function and structure. Prog Biophys Mol Biol. 1985;45(3):149–195. doi: 10.1016/0079-6107(85)90001-x. [DOI] [PubMed] [Google Scholar]
  15. Hultquist D. E., Moyer R. W., Boyer P. D. The preparation and characterization of 1-phosphohistidine and 3-phosphohistidine. Biochemistry. 1966 Jan;5(1):322–331. doi: 10.1021/bi00865a041. [DOI] [PubMed] [Google Scholar]
  16. Ikeda-Saito M., Iizuka T., Yamamoto H., Kayne F. J., Yonetani T. Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen. J Biol Chem. 1977 Jul 25;252(14):4882–4887. [PubMed] [Google Scholar]
  17. Lodi P. J., Knowles J. R. Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications. Biochemistry. 1991 Jul 16;30(28):6948–6956. doi: 10.1021/bi00242a020. [DOI] [PubMed] [Google Scholar]
  18. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  19. Markley J. L., Finkenstadt W. R. Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. III. Mutual electrostatic interaction between histidine residues 12 and 119. Biochemistry. 1975 Aug 12;14(16):3562–3566. doi: 10.1021/bi00687a008. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Meadow N. D., Roseman S. Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a glucose-specific phosphocarrier protein (IIIGlc) from Salmonella typhimurium. J Biol Chem. 1982 Dec 10;257(23):14526–14537. [PubMed] [Google Scholar]
  22. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pelton J. G., Torchia D. A., Meadow N. D., Roseman S. Structural comparison of phosphorylated and unphosphorylated forms of IIIGlc, a signal-transducing protein from Escherichia coli, using three-dimensional NMR techniques. Biochemistry. 1992 Jun 9;31(22):5215–5224. doi: 10.1021/bi00137a017. [DOI] [PubMed] [Google Scholar]
  24. Pelton J. G., Torchia D. A., Meadow N. D., Wong C. Y., Roseman S. Secondary structure of the phosphocarrier protein IIIGlc, a signal-transducing protein from Escherichia coli, determined by heteronuclear three-dimensional NMR spectroscopy. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3479–3483. doi: 10.1073/pnas.88.8.3479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Perutz M. F., Gronenborn A. M., Clore G. M., Fogg J. H., Shih D. T. The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices. J Mol Biol. 1985 Jun 5;183(3):491–498. doi: 10.1016/0022-2836(85)90016-6. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Presper K. A., Wong C. Y., Liu L., Meadow N. D., Roseman S. Site-directed mutagenesis of the phosphocarrier protein. IIIGlc, a major signal-transducing protein in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4052–4055. doi: 10.1073/pnas.86.11.4052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reynolds W. F., Peat I. R., Freedman M. H., Lyerla J. R., Jr Determination of the tautomeric form of the imidazole ring of L-histidine in basic solution by carbon-13 magnetic resonance spectroscopy. J Am Chem Soc. 1973 Jan 24;95(2):328–331. doi: 10.1021/ja00783a006. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Sali D., Bycroft M., Fersht A. R. Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain. Nature. 1988 Oct 20;335(6192):740–743. doi: 10.1038/335740a0. [DOI] [PubMed] [Google Scholar]
  31. Sharma S., Georges F., Delbaere L. T., Lee J. S., Klevit R. E., Waygood E. B. Epitope mapping by mutagenesis distinguishes between the two tertiary structures of the histidine-containing protein HPr. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4877–4881. doi: 10.1073/pnas.88.11.4877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  33. Van Dijk A. A., Scheek R. M., Dijkstra K., Wolters G. K., Robillard G. T. Characterization of the protonation and hydrogen bonding state of the histidine residues in IIAmtl, a domain of the phosphoenolpyruvate-dependent mannitol-specific transport protein. Biochemistry. 1992 Sep 22;31(37):9063–9072. doi: 10.1021/bi00152a050. [DOI] [PubMed] [Google Scholar]
  34. Wang J. F., Hinck A. P., Loh S. N., Markley J. L. Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex. Biochemistry. 1990 Jan 9;29(1):102–113. doi: 10.1021/bi00453a012. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Weigel N., Waygood E. B., Kukuruzinska M. A., Nakazawa A., Roseman S. Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of enzyme I from Salmonella typhimurium. J Biol Chem. 1982 Dec 10;257(23):14461–14469. [PubMed] [Google Scholar]
  37. Wittekind M., Rajagopal P., Branchini B. R., Reizer J., Saier M. H., Jr, Klevit R. E. Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy. Protein Sci. 1992 Oct;1(10):1363–1376. doi: 10.1002/pro.5560011016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Worthylake D., Meadow N. D., Roseman S., Liao D. I., Herzberg O., Remington S. J. Three-dimensional structure of the Escherichia coli phosphocarrier protein IIIglc. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10382–10386. doi: 10.1073/pnas.88.23.10382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. van Dijk A. A., de Lange L. C., Bachovchin W. W., Robillard G. T. Effect of phosphorylation on hydrogen-bonding interactions of the active site histidine of the phosphocarrier protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system determined by 15N NMR spectroscopy. Biochemistry. 1990 Sep 4;29(35):8164–8171. doi: 10.1021/bi00487a026. [DOI] [PubMed] [Google Scholar]

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