Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 Apr;9(4):799–811. doi: 10.1110/ps.9.4.799

Folding of a dimeric beta-barrel: residual structure in the urea denatured state of the human papillomavirus E2 DNA binding domain.

Y K Mok 1, L G Alonso 1, L M Lima 1, M Bycroft 1, G de Prat-Gay 1
PMCID: PMC2144607  PMID: 10794423

Abstract

The dimeric beta-barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papillomaviruses. We have previously described the kinetic folding mechanism of the human HPV-16 domain, and, as part of these studies, we present a structural characterization of the urea-denatured state of the protein. We have obtained a set of chemical shift assignments for the C-terminal domain in urea using heteronuclear NMR methods and found regions with persistent residual structure. Based on chemical shift deviations from random coil values, 3'J(NHN alpha) coupling constants, heteronuclear single quantum coherence peak intensities, and nuclear Overhauser effect data, we have determined clusters of residual structure in regions corresponding to the DNA binding helix and the second beta-strand in the folded conformation. Most of the structures found are of nonnative nature, including turn-like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration dependence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and the rate-limiting step becomes first order at high protein concentrations. The structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a better understanding of the folding of dimers in general and intertwined dimeric proteins such as transcription factors in particular.

Full Text

The Full Text of this article is available as a PDF (661.2 KB).

Selected References

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

  1. Arcus V. L., Vuilleumier S., Freund S. M., Bycroft M., Fersht A. R. A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding. J Mol Biol. 1995 Nov 24;254(2):305–321. doi: 10.1006/jmbi.1995.0618. [DOI] [PubMed] [Google Scholar]
  2. Arcus V. L., Vuilleumier S., Freund S. M., Bycroft M., Fersht A. R. Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9412–9416. doi: 10.1073/pnas.91.20.9412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bochkarev A., Barwell J. A., Pfuetzner R. A., Bochkareva E., Frappier L., Edwards A. M. Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein, EBNA1, bound to DNA. Cell. 1996 Mar 8;84(5):791–800. doi: 10.1016/s0092-8674(00)81056-9. [DOI] [PubMed] [Google Scholar]
  4. Buck M., Schwalbe H., Dobson C. M. Characterization of conformational preferences in a partly folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol. Biochemistry. 1995 Oct 10;34(40):13219–13232. doi: 10.1021/bi00040a038. [DOI] [PubMed] [Google Scholar]
  5. Edwards A. M., Bochkarev A., Frappier L. Origin DNA-binding proteins. Curr Opin Struct Biol. 1998 Feb;8(1):49–53. doi: 10.1016/s0959-440x(98)80009-2. [DOI] [PubMed] [Google Scholar]
  6. Evans P. A., Topping K. D., Woolfson D. N., Dobson C. M. Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme. Proteins. 1991;9(4):248–266. doi: 10.1002/prot.340090404. [DOI] [PubMed] [Google Scholar]
  7. Foguel D., Silva J. L., de Prat-Gay G. Characterization of a partially folded monomer of the DNA-binding domain of human papillomavirus E2 protein obtained at high pressure. J Biol Chem. 1998 Apr 10;273(15):9050–9057. doi: 10.1074/jbc.273.15.9050. [DOI] [PubMed] [Google Scholar]
  8. Grzesiek S., Bax A. Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomol NMR. 1993 Mar;3(2):185–204. doi: 10.1007/BF00178261. [DOI] [PubMed] [Google Scholar]
  9. Hawley-Nelson P., Androphy E. J., Lowy D. R., Schiller J. T. The specific DNA recognition sequence of the bovine papillomavirus E2 protein is an E2-dependent enhancer. EMBO J. 1988 Feb;7(2):525–531. doi: 10.1002/j.1460-2075.1988.tb02841.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hegde R. S., Androphy E. J. Crystal structure of the E2 DNA-binding domain from human papillomavirus type 16: implications for its DNA binding-site selection mechanism. J Mol Biol. 1998 Dec 18;284(5):1479–1489. doi: 10.1006/jmbi.1998.2260. [DOI] [PubMed] [Google Scholar]
  11. Hegde R. S., Grossman S. R., Laimins L. A., Sigler P. B. Crystal structure at 1.7 A of the bovine papillomavirus-1 E2 DNA-binding domain bound to its DNA target. Nature. 1992 Oct 8;359(6395):505–512. doi: 10.1038/359505a0. [DOI] [PubMed] [Google Scholar]
  12. Hegde R. S., Wang A. F., Kim S. S., Schapira M. Subunit rearrangement accompanies sequence-specific DNA binding by the bovine papillomavirus-1 E2 protein. J Mol Biol. 1998 Mar 6;276(4):797–808. doi: 10.1006/jmbi.1997.1587. [DOI] [PubMed] [Google Scholar]
  13. Liang H., Petros A. M., Meadows R. P., Yoon H. S., Egan D. A., Walter K., Holzman T. F., Robins T., Fesik S. W. Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions. Biochemistry. 1996 Feb 20;35(7):2095–2103. doi: 10.1021/bi951932w. [DOI] [PubMed] [Google Scholar]
  14. Lima L. M., de Prat-Gay G. Conformational changes and stabilization induced by ligand binding in the DNA-binding domain of the E2 protein from human papillomavirus. J Biol Chem. 1997 Aug 1;272(31):19295–19303. doi: 10.1074/jbc.272.31.19295. [DOI] [PubMed] [Google Scholar]
  15. Logan T. M., Thériault Y., Fesik S. W. Structural characterization of the FK506 binding protein unfolded in urea and guanidine hydrochloride. J Mol Biol. 1994 Feb 18;236(2):637–648. doi: 10.1006/jmbi.1994.1173. [DOI] [PubMed] [Google Scholar]
  16. Marion D., Driscoll P. C., Kay L. E., Wingfield P. T., Bax A., Gronenborn A. M., Clore G. M. Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta. Biochemistry. 1989 Jul 25;28(15):6150–6156. doi: 10.1021/bi00441a004. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. McBride A. A., Byrne J. C., Howley P. M. E2 polypeptides encoded by bovine papillomavirus type 1 form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain. Proc Natl Acad Sci U S A. 1989 Jan;86(2):510–514. doi: 10.1073/pnas.86.2.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Merutka G., Dyson H. J., Wright P. E. 'Random coil' 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG. J Biomol NMR. 1995 Jan;5(1):14–24. doi: 10.1007/BF00227466. [DOI] [PubMed] [Google Scholar]
  20. Mok Y. K., Bycroft M., de Prat-Gay G. The dimeric DNA binding domain of the human papillomavirus E2 protein folds through a monomeric intermediate which cannot be native-like. Nat Struct Biol. 1996 Aug;3(8):711–717. doi: 10.1038/nsb0896-711. [DOI] [PubMed] [Google Scholar]
  21. Mok Y. K., de Prat Gay G., Butler P. J., Bycroft M. Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain. Protein Sci. 1996 Feb;5(2):310–319. doi: 10.1002/pro.5560050215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neri D., Billeter M., Wider G., Wüthrich K. NMR determination of residual structure in a urea-denatured protein, the 434-repressor. Science. 1992 Sep 11;257(5076):1559–1563. doi: 10.1126/science.1523410. [DOI] [PubMed] [Google Scholar]
  23. Neri D., Wider G., Wüthrich K. 1H, 15N and 13C NMR assignments of the 434 repressor fragments 1-63 and 44-63 unfolded in 7 M urea. FEBS Lett. 1992 Jun 1;303(2-3):129–135. doi: 10.1016/0014-5793(92)80504-a. [DOI] [PubMed] [Google Scholar]
  24. Neri D., Wider G., Wüthrich K. Complete 15N and 1H NMR assignments for the amino-terminal domain of the phage 434 repressor in the urea-unfolded form. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4397–4401. doi: 10.1073/pnas.89.10.4397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith L. J., Bolin K. A., Schwalbe H., MacArthur M. W., Thornton J. M., Dobson C. M. Analysis of main chain torsion angles in proteins: prediction of NMR coupling constants for native and random coil conformations. J Mol Biol. 1996 Jan 26;255(3):494–506. doi: 10.1006/jmbi.1996.0041. [DOI] [PubMed] [Google Scholar]
  26. Swindells M. B., MacArthur M. W., Thornton J. M. Intrinsic phi, psi propensities of amino acids, derived from the coil regions of known structures. Nat Struct Biol. 1995 Jul;2(7):596–603. doi: 10.1038/nsb0795-596. [DOI] [PubMed] [Google Scholar]
  27. Wishart D. S., Bigam C. G., Holm A., Hodges R. S., Sykes B. D. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR. 1995 Jan;5(1):67–81. doi: 10.1007/BF00227471. [DOI] [PubMed] [Google Scholar]
  28. Wishart D. S., Sykes B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR. 1994 Mar;4(2):171–180. doi: 10.1007/BF00175245. [DOI] [PubMed] [Google Scholar]
  29. Yao J., Dyson H. J., Wright P. E. Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins. FEBS Lett. 1997 Dec 15;419(2-3):285–289. doi: 10.1016/s0014-5793(97)01474-9. [DOI] [PubMed] [Google Scholar]
  30. Zhang O., Forman-Kay J. D., Shortle D., Kay L. E. Triple-resonance NOESY-based experiments with improved spectral resolution: applications to structural characterization of unfolded, partially folded and folded proteins. J Biomol NMR. 1997 Feb;9(2):181–200. doi: 10.1023/a:1018658305040. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES