Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 Oct;80(19):5837–5841. doi: 10.1073/pnas.80.19.5837

Secondary structure of the lac repressor DNA-binding domain by two-dimensional 1H nuclear magnetic resonance in solution.

E R Zuiderweg, R Kaptein, K Wüthrich
PMCID: PMC390170  PMID: 6351066

Abstract

A recently proposed approach for spatial structure determination in noncrystalline proteins by nuclear magnetic resonance was applied to the lac repressor DNA-binding domain. On the basis of sequence-specific 1H NMR assignments, the location of alpha-helices in the amino acid sequence was determined from nuclear Overhauser enhancement data and from amide proton exchange studies. These investigations provide detailed experimental data on the structure of a noncrystalline DNA-binding protein. The results support the hypothesis advanced by others that sequence-specific interactions between lac repressor and DNA are mediated by a particular spatial arrangement of two alpha-helices common to various different DNA-binding proteins.

Full text

PDF
5837

Images in this article

5839Image
on p.5839

Selected References

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

  1. Anderson W. F., Ohlendorf D. H., Takeda Y., Matthews B. W. Structure of the cro repressor from bacteriophage lambda and its interaction with DNA. Nature. 1981 Apr 30;290(5809):754–758. doi: 10.1038/290754a0. [DOI] [PubMed] [Google Scholar]
  2. Arndt K. T., Boschelli F., Lu P., Miller J. H. lac Repressor: a proton magnetic resonance look at the deoxyribonucleic acid binding fragment. Biochemistry. 1981 Oct 13;20(21):6109–6118. doi: 10.1021/bi00524a030. [DOI] [PubMed] [Google Scholar]
  3. Arndt K., Nick H., Boschelli F., Lu P., Sadler J. Repressor--operator interaction in the lac operon. III. Nuclear magnetic resonance observations with altered amino-terminal DNA binding domains. J Mol Biol. 1982 Nov 5;161(3):439–457. doi: 10.1016/0022-2836(82)90248-0. [DOI] [PubMed] [Google Scholar]
  4. Arseniev A. S., Wider G., Joubert F. J., Wüthrich K. Assignment of the H nuclear magnetic resonance spectrum of the trypsin inhibitor E from Dendroaspis polylepis polylepis Two-dimensional nuclear magnetic resonance at 500 MHz. J Mol Biol. 1982 Aug 5;159(2):323–351. doi: 10.1016/0022-2836(82)90498-3. [DOI] [PubMed] [Google Scholar]
  5. Billeter M., Braun W., Wüthrich K. Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations. J Mol Biol. 1982 Mar 5;155(3):321–346. doi: 10.1016/0022-2836(82)90008-0. [DOI] [PubMed] [Google Scholar]
  6. Bourgeois S., Pfahl M. Repressors. Adv Protein Chem. 1976;30:1–99. doi: 10.1016/s0065-3233(08)60478-7. [DOI] [PubMed] [Google Scholar]
  7. Braun W., Bösch C., Brown L. R., Go N., Wüthrich K. Combined use of proton-proton Overhauser enhancements and a distance geometry algorithm for determination of polypeptide conformations. Application to micelle-bound glucagon. Biochim Biophys Acta. 1981 Feb 27;667(2):377–396. doi: 10.1016/0005-2795(81)90205-1. [DOI] [PubMed] [Google Scholar]
  8. Buck F., Rüterjans H., Beyreuther K. 1H NMR study of the lactose repressor from Escherichia coli. FEBS Lett. 1978 Dec 15;96(2):335–338. doi: 10.1016/0014-5793(78)80430-x. [DOI] [PubMed] [Google Scholar]
  9. Buck F., Rüterjans H., Kaptein R., Beyreuther K. Photochemically induced dynamic nuclear polarization investigation of complex formation of the NH2-terminal DNA-binding domain of lac repressor with poly[d(AT)]. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5145–5148. doi: 10.1073/pnas.77.9.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Geisler N., Weber K. Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding. Biochemistry. 1977 Mar 8;16(5):938–943. doi: 10.1021/bi00624a020. [DOI] [PubMed] [Google Scholar]
  11. Hvidt A., Nielsen S. O. Hydrogen exchange in proteins. Adv Protein Chem. 1966;21:287–386. doi: 10.1016/s0065-3233(08)60129-1. [DOI] [PubMed] [Google Scholar]
  12. Matthews B. W., Ohlendorf D. H., Anderson W. F., Takeda Y. Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1428–1432. doi: 10.1073/pnas.79.5.1428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. McKay D. B., Steitz T. A. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981 Apr 30;290(5809):744–749. doi: 10.1038/290744a0. [DOI] [PubMed] [Google Scholar]
  14. Miller J. H., Coulondre C., Hofer M., Schmeissner U., Sommer H., Schmitz A., Lu P. Genetic studies of the lac repressor. IX. Generation of altered proteins by the suppression of nonsence mutations. J Mol Biol. 1979 Jun 25;131(2):191–222. doi: 10.1016/0022-2836(79)90073-1. [DOI] [PubMed] [Google Scholar]
  15. Nagayama K. Two-dimensional NMR spectroscopy: an application to the study of flexibility of protein molecules. Adv Biophys. 1981;14:139–204. [PubMed] [Google Scholar]
  16. Nick H., Arndt K., Boschelli F., Jarema M. A., Lillis M., Sadler J., Caruthers M., Lu P. lac repressor-lac operator interaction: NMR observations. Proc Natl Acad Sci U S A. 1982 Jan;79(2):218–222. doi: 10.1073/pnas.79.2.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nick H., Arndt K., Boschelli F., Jarema M. A., Lillis M., Sommer H., Lu P., Sadler J. Repressor--operator interaction in the lac operon. II. Observations at the tyrosines and tryptophans. J Mol Biol. 1982 Nov 5;161(3):417–438. doi: 10.1016/0022-2836(82)90247-9. [DOI] [PubMed] [Google Scholar]
  18. Ogata R. T., Gilbert W. DNA-binding site of lac repressor probed by dimethylsulfate methylation of lac operator. J Mol Biol. 1979 Aug 25;132(4):709–728. doi: 10.1016/0022-2836(79)90384-x. [DOI] [PubMed] [Google Scholar]
  19. Ohlendorf D. H., Anderson W. F., Fisher R. G., Takeda Y., Matthews B. W. The molecular basis of DNA-protein recognition inferred from the structure of cro repressor. Nature. 1982 Aug 19;298(5876):718–723. doi: 10.1038/298718a0. [DOI] [PubMed] [Google Scholar]
  20. Pabo C. O., Lewis M. The operator-binding domain of lambda repressor: structure and DNA recognition. Nature. 1982 Jul 29;298(5873):443–447. doi: 10.1038/298443a0. [DOI] [PubMed] [Google Scholar]
  21. Ribeiro A. A., Wemmer D., Bray R. P., Jardetzky O. A folded structure for the lac-repressor headpiece. Biochem Biophys Res Commun. 1981 Mar 31;99(2):668–674. doi: 10.1016/0006-291x(81)91796-4. [DOI] [PubMed] [Google Scholar]
  22. Ribeiro A. A., Wemmer D., Bray R. P., Jardetzky O. pH dependence of the high-resolution proton nuclear magnetic resonance spectrum of the lac repressor headpiece. Biochemistry. 1981 Jun 9;20(12):3346–3350. doi: 10.1021/bi00515a005. [DOI] [PubMed] [Google Scholar]
  23. Ribeiro A. A., Wemmer D., Bray R. P., Wade-Jardetzky N. G., Jardetzky O. High-resolution nuclear magnetic resonance studies of the Lac repressor. 1. Assignments of tyrosine resonances in the N-terminal headpiece. Biochemistry. 1981 Feb 17;20(4):818–823. doi: 10.1021/bi00507a025. [DOI] [PubMed] [Google Scholar]
  24. Ribeiro A. A., Wemmer D., Bray R. P., Wade-Jardetzky N. G., Jardetzky O. High-resolution nuclear magnetic resonance studies of the Lac repressor. 2. Partial analysis of the aliphatic region of the Lac repressor headpiece spectrum. Biochemistry. 1981 Feb 17;20(4):823–829. doi: 10.1021/bi00507a026. [DOI] [PubMed] [Google Scholar]
  25. Sauer R. T., Yocum R. R., Doolittle R. F., Lewis M., Pabo C. O. Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature. 1982 Jul 29;298(5873):447–451. doi: 10.1038/298447a0. [DOI] [PubMed] [Google Scholar]
  26. Scheek R. M., Zuiderweg E. R., Klappe K. J., van Boom J. H., Kaptein R., Rüterjans H., Beyreuther K. lac Repressor headpiece binds specifically to half of the lac operator: a proton nuclear magnetic resonance study. Biochemistry. 1983 Jan 4;22(1):228–235. doi: 10.1021/bi00270a033. [DOI] [PubMed] [Google Scholar]
  27. Steitz T. A., Ohlendorf D. H., McKay D. B., Anderson W. F., Matthews B. W. Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins. Proc Natl Acad Sci U S A. 1982 May;79(10):3097–3100. doi: 10.1073/pnas.79.10.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wade-Jardetzky N., Bray R. P., Conover W. W., Jardetzky O., Geisler N., Weber K. Differential mobility of the N-terminal headpiece in the lac-repressor protein. J Mol Biol. 1979 Feb 25;128(2):259–264. doi: 10.1016/0022-2836(79)90129-3. [DOI] [PubMed] [Google Scholar]
  29. Wagner G., Wüthrich K. Amide protein exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution. Studies with two-dimensional nuclear magnetic resonance. J Mol Biol. 1982 Sep 15;160(2):343–361. doi: 10.1016/0022-2836(82)90180-2. [DOI] [PubMed] [Google Scholar]
  30. Wagner G., Wüthrich K. Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Basic pancreatic trypsin inhibitor. J Mol Biol. 1982 Mar 5;155(3):347–366. doi: 10.1016/0022-2836(82)90009-2. [DOI] [PubMed] [Google Scholar]
  31. Weber I. T., McKay D. B., Steitz T. A. Two helix DNA binding motif of CAP found in lac repressor and gal repressor. Nucleic Acids Res. 1982 Aug 25;10(16):5085–5102. doi: 10.1093/nar/10.16.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wemmer D., Ribeiro A. A., Bray R. P., Wade-Jardetzky N. G., Jardetzky O. High-resolution nuclear magnetic resonance studies of the Lac repressor. 3. Unfolding of the Lac repressor headpiece. Biochemistry. 1981 Feb 17;20(4):829–833. doi: 10.1021/bi00507a027. [DOI] [PubMed] [Google Scholar]
  33. Wemmer D., Shvo H., Ribeiro A. A., Bray R. P., Jardetzky O. High-resolution proton nuclear magnetic resonance studies of the exchangeable resonances of the lac repressor headpiece. Biochemistry. 1981 Jun 9;20(12):3351–3354. doi: 10.1021/bi00515a006. [DOI] [PubMed] [Google Scholar]
  34. Wider G., Lee K. H., Wüthrich K. Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Glucagon bound to perdeuterated dodecylphosphocholine micelles. J Mol Biol. 1982 Mar 5;155(3):367–388. doi: 10.1016/0022-2836(82)90010-9. [DOI] [PubMed] [Google Scholar]
  35. Wüthrich K. Sequential individual resonance assignments in the 1H-nmr spectra of polypeptides and proteins. Biopolymers. 1983 Jan;22(1):131–138. doi: 10.1002/bip.360220121. [DOI] [PubMed] [Google Scholar]
  36. Wüthrich K., Wider G., Wagner G., Braun W. Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance. J Mol Biol. 1982 Mar 5;155(3):311–319. doi: 10.1016/0022-2836(82)90007-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES