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. 1992 Nov;1(11):1403–1412. doi: 10.1002/pro.5560011102

Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA-binding domain as studied by amide proton exchange.

T Mau 1, J D Baleja 1, G Wagner 1
PMCID: PMC2142116  PMID: 1303761

Abstract

Backbone amide proton exchange rates in the DNA-binding domain of GAL4 have been determined using 1H-15N heteronuclear correlation NMR spectroscopy. Three forms of the protein were studied-the native Zn-containing protein, the Cd-substituted protein, and a Zn-GAL4/DNA complex. Exchange rates in the Zn-containing protein are significantly slower than in the Cd-substituted protein. This shows that Cd-substituted GAL4 is destabilized relative to the native Zn-containing protein. Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA-recognition module are significantly reduced by the presence of DNA. In all forms of the protein, the internal dyad symmetry of the DNA-recognition module of GAL4 is reflected by the backbone amide proton exchange rates.

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Selected References

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

  1. Baleja J. D., Marmorstein R., Harrison S. C., Wagner G. Solution structure of the DNA-binding domain of Cd2-GAL4 from S. cerevisiae. Nature. 1992 Apr 2;356(6368):450–453. doi: 10.1038/356450a0. [DOI] [PubMed] [Google Scholar]
  2. Bram R. J., Kornberg R. D. Specific protein binding to far upstream activating sequences in polymerase II promoters. Proc Natl Acad Sci U S A. 1985 Jan;82(1):43–47. doi: 10.1073/pnas.82.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carey M., Kakidani H., Leatherwood J., Mostashari F., Ptashne M. An amino-terminal fragment of GAL4 binds DNA as a dimer. J Mol Biol. 1989 Oct 5;209(3):423–432. doi: 10.1016/0022-2836(89)90007-7. [DOI] [PubMed] [Google Scholar]
  4. Englander S. W., Downer N. W., Teitelbaum H. Hydrogen exchange. Annu Rev Biochem. 1972;41:903–924. doi: 10.1146/annurev.bi.41.070172.004351. [DOI] [PubMed] [Google Scholar]
  5. Englander S. W., Kallenbach N. R. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q Rev Biophys. 1983 Nov;16(4):521–655. doi: 10.1017/s0033583500005217. [DOI] [PubMed] [Google Scholar]
  6. Gadhavi P. L., Davis A. L., Povey J. F., Keeler J., Laue E. D. Polypeptide-metal cluster connectivities in Cd(II) GAL4. FEBS Lett. 1991 Apr 9;281(1-2):223–226. doi: 10.1016/0014-5793(91)80398-m. [DOI] [PubMed] [Google Scholar]
  7. Gadhavi P. L., Raine A. R., Alefounder P. R., Laue E. D. Complete assignment of the 1H NMR spectrum and secondary structure of the DNA binding domain of GAL4. FEBS Lett. 1990 Dec 10;276(1-2):49–53. doi: 10.1016/0014-5793(90)80504-c. [DOI] [PubMed] [Google Scholar]
  8. Gardner K. H., Pan T., Narula S., Rivera E., Coleman J. E. Structure of the binuclear metal-binding site in the GAL4 transcription factor. Biochemistry. 1991 Nov 26;30(47):11292–11302. doi: 10.1021/bi00111a015. [DOI] [PubMed] [Google Scholar]
  9. Giniger E., Varnum S. M., Ptashne M. Specific DNA binding of GAL4, a positive regulatory protein of yeast. Cell. 1985 Apr;40(4):767–774. doi: 10.1016/0092-8674(85)90336-8. [DOI] [PubMed] [Google Scholar]
  10. Harrison S. C. A structural taxonomy of DNA-binding domains. Nature. 1991 Oct 24;353(6346):715–719. doi: 10.1038/353715a0. [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. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Keating K. M., Ghosaini L. R., Giedroc D. P., Williams K. R., Coleman J. E., Sturtevant J. M. Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution. Biochemistry. 1988 Jul 12;27(14):5240–5245. doi: 10.1021/bi00414a044. [DOI] [PubMed] [Google Scholar]
  14. Kraulis P. J., Raine A. R., Gadhavi P. L., Laue E. D. Structure of the DNA-binding domain of zinc GAL4. Nature. 1992 Apr 2;356(6368):448–450. doi: 10.1038/356448a0. [DOI] [PubMed] [Google Scholar]
  15. Marmorstein R., Carey M., Ptashne M., Harrison S. C. DNA recognition by GAL4: structure of a protein-DNA complex. Nature. 1992 Apr 2;356(6368):408–414. doi: 10.1038/356408a0. [DOI] [PubMed] [Google Scholar]
  16. Messerle B. A., Schäffer A., Vasák M., Kägi J. H., Wüthrich K. Comparison of the solution conformations of human [Zn7]-metallothionein-2 and [Cd7]-metallothionein-2 using nuclear magnetic resonance spectroscopy. J Mol Biol. 1992 May 20;225(2):433–443. doi: 10.1016/0022-2836(92)90930-i. [DOI] [PubMed] [Google Scholar]
  17. Pan T., Coleman J. E. GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2077–2081. doi: 10.1073/pnas.87.6.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pan T., Coleman J. E. Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor. Proc Natl Acad Sci U S A. 1989 May;86(9):3145–3149. doi: 10.1073/pnas.86.9.3145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Paterson Y., Englander S. W., Roder H. An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR. Science. 1990 Aug 17;249(4970):755–759. doi: 10.1126/science.1697101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Povey J. F., Diakun G. P., Garner C. D., Wilson S. P., Laue E. D. Metal ion co-ordination in the DNA binding domain of the yeast transcriptional activator GAL4. FEBS Lett. 1990 Jun 18;266(1-2):142–146. doi: 10.1016/0014-5793(90)81525-s. [DOI] [PubMed] [Google Scholar]
  21. Richarz R., Sehr P., Wagner G., Wüthrich K. Kinetics of the exchange of individual amide protons in the basic pancreatic trypsin inhibitor. J Mol Biol. 1979 May 5;130(1):19–30. doi: 10.1016/0022-2836(79)90549-7. [DOI] [PubMed] [Google Scholar]
  22. Takahashi H., Odaka A., Kawaminami S., Matsunaga C., Kato K., Shimada I., Arata Y. Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody. Biochemistry. 1991 Jul 2;30(26):6611–6619. doi: 10.1021/bi00240a034. [DOI] [PubMed] [Google Scholar]
  23. Wagner G. Characterization of the distribution of internal motions in the basic pancreatic trypsin inhibitor using a large number of internal NMR probes. Q Rev Biophys. 1983 Feb;16(1):1–57. doi: 10.1017/s0033583500004911. [DOI] [PubMed] [Google Scholar]
  24. 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]

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