Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1999 May 4;18(9):2563–2579. doi: 10.1093/emboj/18.9.2563

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding.

F H Allain 1, Y M Yen 1, J E Masse 1, P Schultze 1, T Dieckmann 1, R C Johnson 1, J Feigon 1
PMCID: PMC1171337  PMID: 10228169

Abstract

NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Balaeff A., Churchill M. E., Schulten K. Structure prediction of a complex between the chromosomal protein HMG-D and DNA. Proteins. 1998 Feb 1;30(2):113–135. doi: 10.1002/(sici)1097-0134(19980201)30:2<113::aid-prot2>3.0.co;2-o. [DOI] [PubMed] [Google Scholar]
  2. Bustin M., Reeves R. High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function. Prog Nucleic Acid Res Mol Biol. 1996;54:35–100. doi: 10.1016/s0079-6603(08)60360-8. [DOI] [PubMed] [Google Scholar]
  3. Churchill M. E., Jones D. N., Glaser T., Hefner H., Searles M. A., Travers A. A. HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG. EMBO J. 1995 Mar 15;14(6):1264–1275. doi: 10.1002/j.1460-2075.1995.tb07110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Costigan C., Kolodrubetz D., Snyder M. NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway. Mol Cell Biol. 1994 Apr;14(4):2391–2403. doi: 10.1128/mcb.14.4.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Czisch M., Boelens R. Sensitivity enhancement in the TROSY experiment. J Magn Reson. 1998 Sep;134(1):158–160. doi: 10.1006/jmre.1998.1483. [DOI] [PubMed] [Google Scholar]
  6. Dornberger U., Flemming J., Fritzsche H. Structure determination and analysis of helix parameters in the DNA decamer d(CATGGCCATG)2 comparison of results from NMR and crystallography. J Mol Biol. 1998 Dec 18;284(5):1453–1463. doi: 10.1006/jmbi.1998.2261. [DOI] [PubMed] [Google Scholar]
  7. Ellenberger T., Fass D., Arnaud M., Harrison S. C. Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer. Genes Dev. 1994 Apr 15;8(8):970–980. doi: 10.1101/gad.8.8.970. [DOI] [PubMed] [Google Scholar]
  8. Ge H., Roeder R. G. The high mobility group protein HMG1 can reversibly inhibit class II gene transcription by interaction with the TATA-binding protein. J Biol Chem. 1994 Jun 24;269(25):17136–17140. [PubMed] [Google Scholar]
  9. Grosschedl R., Giese K., Pagel J. HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 1994 Mar;10(3):94–100. doi: 10.1016/0168-9525(94)90232-1. [DOI] [PubMed] [Google Scholar]
  10. Gubbay J., Collignon J., Koopman P., Capel B., Economou A., Münsterberg A., Vivian N., Goodfellow P., Lovell-Badge R. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature. 1990 Jul 19;346(6281):245–250. doi: 10.1038/346245a0. [DOI] [PubMed] [Google Scholar]
  11. Güntert P., Braun W., Wüthrich K. Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J Mol Biol. 1991 Feb 5;217(3):517–530. doi: 10.1016/0022-2836(91)90754-t. [DOI] [PubMed] [Google Scholar]
  12. Güntert P., Mumenthaler C., Wüthrich K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J Mol Biol. 1997 Oct 17;273(1):283–298. doi: 10.1006/jmbi.1997.1284. [DOI] [PubMed] [Google Scholar]
  13. Haqq C. M., King C. Y., Ukiyama E., Falsafi S., Haqq T. N., Donahoe P. K., Weiss M. A. Molecular basis of mammalian sexual determination: activation of Müllerian inhibiting substance gene expression by SRY. Science. 1994 Dec 2;266(5190):1494–1500. doi: 10.1126/science.7985018. [DOI] [PubMed] [Google Scholar]
  14. Hardman C. H., Broadhurst R. W., Raine A. R., Grasser K. D., Thomas J. O., Laue E. D. Structure of the A-domain of HMG1 and its interaction with DNA as studied by heteronuclear three- and four-dimensional NMR spectroscopy. Biochemistry. 1995 Dec 26;34(51):16596–16607. doi: 10.1021/bi00051a007. [DOI] [PubMed] [Google Scholar]
  15. Higgins D. G., Thompson J. D., Gibson T. J. Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 1996;266:383–402. doi: 10.1016/s0076-6879(96)66024-8. [DOI] [PubMed] [Google Scholar]
  16. Hud N. V., Milanovich F. P., Balhorn R. Evidence of novel secondary structure in DNA-bound protamine is revealed by Raman spectroscopy. Biochemistry. 1994 Jun 21;33(24):7528–7535. doi: 10.1021/bi00190a005. [DOI] [PubMed] [Google Scholar]
  17. Jayaraman L., Moorthy N. C., Murthy K. G., Manley J. L., Bustin M., Prives C. High mobility group protein-1 (HMG-1) is a unique activator of p53. Genes Dev. 1998 Feb 15;12(4):462–472. doi: 10.1101/gad.12.4.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones D. N., Searles M. A., Shaw G. L., Churchill M. E., Ner S. S., Keeler J., Travers A. A., Neuhaus D. The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster. Structure. 1994 Jul 15;2(7):609–627. doi: 10.1016/s0969-2126(00)00063-0. [DOI] [PubMed] [Google Scholar]
  19. Jordan S. R., Pabo C. O. Structure of the lambda complex at 2.5 A resolution: details of the repressor-operator interactions. Science. 1988 Nov 11;242(4880):893–899. doi: 10.1126/science.3187530. [DOI] [PubMed] [Google Scholar]
  20. Kane S. A., Lippard S. J. Photoreactivity of platinum(II) in cisplatin-modified DNA affords specific cross-links to HMG domain proteins. Biochemistry. 1996 Feb 20;35(7):2180–2188. doi: 10.1021/bi952240a. [DOI] [PubMed] [Google Scholar]
  21. Kim J. L., Nikolov D. B., Burley S. K. Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature. 1993 Oct 7;365(6446):520–527. doi: 10.1038/365520a0. [DOI] [PubMed] [Google Scholar]
  22. Kim Y., Geiger J. H., Hahn S., Sigler P. B. Crystal structure of a yeast TBP/TATA-box complex. Nature. 1993 Oct 7;365(6446):512–520. doi: 10.1038/365512a0. [DOI] [PubMed] [Google Scholar]
  23. King C. Y., Weiss M. A. The SRY high-mobility-group box recognizes DNA by partial intercalation in the minor groove: a topological mechanism of sequence specificity. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11990–11994. doi: 10.1073/pnas.90.24.11990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kolodrubetz D., Burgum A. Duplicated NHP6 genes of Saccharomyces cerevisiae encode proteins homologous to bovine high mobility group protein 1. J Biol Chem. 1990 Feb 25;265(6):3234–3239. [PubMed] [Google Scholar]
  25. Koradi R., Billeter M., Wüthrich K. MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph. 1996 Feb;14(1):51-5, 29-32. doi: 10.1016/0263-7855(96)00009-4. [DOI] [PubMed] [Google Scholar]
  26. Kuehl L., Salmond B., Tran L. Concentrations of high-mobility-group proteins in the nucleus and cytoplasm of several rat tissues. J Cell Biol. 1984 Aug;99(2):648–654. doi: 10.1083/jcb.99.2.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Landt O., Grunert H. P., Hahn U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990 Nov 30;96(1):125–128. doi: 10.1016/0378-1119(90)90351-q. [DOI] [PubMed] [Google Scholar]
  28. Laskowski R. A., Rullmannn J. A., MacArthur M. W., Kaptein R., Thornton J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR. 1996 Dec;8(4):477–486. doi: 10.1007/BF00228148. [DOI] [PubMed] [Google Scholar]
  29. Lee W., Revington M. J., Arrowsmith C., Kay L. E. A pulsed field gradient isotope-filtered 3D 13C HMQC-NOESY experiment for extracting intermolecular NOE contacts in molecular complexes. FEBS Lett. 1994 Aug 15;350(1):87–90. doi: 10.1016/0014-5793(94)00740-3. [DOI] [PubMed] [Google Scholar]
  30. Love J. J., Li X., Case D. A., Giese K., Grosschedl R., Wright P. E. Structural basis for DNA bending by the architectural transcription factor LEF-1. Nature. 1995 Aug 31;376(6543):791–795. doi: 10.1038/376791a0. [DOI] [PubMed] [Google Scholar]
  31. Luisi B. F., Xu W. X., Otwinowski Z., Freedman L. P., Yamamoto K. R., Sigler P. B. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991 Aug 8;352(6335):497–505. doi: 10.1038/352497a0. [DOI] [PubMed] [Google Scholar]
  32. Masse J. E., Bortmann P., Dieckmann T., Feigon J. Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies. Nucleic Acids Res. 1998 Jun 1;26(11):2618–2624. doi: 10.1093/nar/26.11.2618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Newman M., Strzelecka T., Dorner L. F., Schildkraut I., Aggarwal A. K. Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding. Science. 1995 Aug 4;269(5224):656–663. doi: 10.1126/science.7624794. [DOI] [PubMed] [Google Scholar]
  34. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  35. Nightingale K., Dimitrov S., Reeves R., Wolffe A. P. Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin. EMBO J. 1996 Feb 1;15(3):548–561. [PMC free article] [PubMed] [Google Scholar]
  36. Paull T. T., Carey M., Johnson R. C. Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro. Genes Dev. 1996 Nov 1;10(21):2769–2781. doi: 10.1101/gad.10.21.2769. [DOI] [PubMed] [Google Scholar]
  37. Paull T. T., Haykinson M. J., Johnson R. C. The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures. Genes Dev. 1993 Aug;7(8):1521–1534. doi: 10.1101/gad.7.8.1521. [DOI] [PubMed] [Google Scholar]
  38. Paull T. T., Johnson R. C. DNA looping by Saccharomyces cerevisiae high mobility group proteins NHP6A/B. Consequences for nucleoprotein complex assembly and chromatin condensation. J Biol Chem. 1995 Apr 14;270(15):8744–8754. doi: 10.1074/jbc.270.15.8744. [DOI] [PubMed] [Google Scholar]
  39. Payet D., Travers A. The acidic tail of the high mobility group protein HMG-D modulates the structural selectivity of DNA binding. J Mol Biol. 1997 Feb 14;266(1):66–75. doi: 10.1006/jmbi.1996.0782. [DOI] [PubMed] [Google Scholar]
  40. Peters R., King C. Y., Ukiyama E., Falsafi S., Donahoe P. K., Weiss M. A. An SRY mutation causing human sex reversal resolves a general mechanism of structure-specific DNA recognition: application to the four-way DNA junction. Biochemistry. 1995 Apr 11;34(14):4569–4576. doi: 10.1021/bi00014a009. [DOI] [PubMed] [Google Scholar]
  41. Pil P. M., Chow C. S., Lippard S. J. High-mobility-group 1 protein mediates DNA bending as determined by ring closures. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9465–9469. doi: 10.1073/pnas.90.20.9465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pil P. M., Lippard S. J. Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin. Science. 1992 Apr 10;256(5054):234–237. doi: 10.1126/science.1566071. [DOI] [PubMed] [Google Scholar]
  43. Read C. M., Cary P. D., Crane-Robinson C., Driscoll P. C., Norman D. G. Solution structure of a DNA-binding domain from HMG1. Nucleic Acids Res. 1993 Jul 25;21(15):3427–3436. doi: 10.1093/nar/21.15.3427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Salzmann M., Pervushin K., Wider G., Senn H., Wüthrich K. TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13585–13590. doi: 10.1073/pnas.95.23.13585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shykind B. M., Kim J., Sharp P. A. Activation of the TFIID-TFIIA complex with HMG-2. Genes Dev. 1995 Jun 1;9(11):1354–1365. doi: 10.1101/gad.9.11.1354. [DOI] [PubMed] [Google Scholar]
  46. Sinclair A. H., Berta P., Palmer M. S., Hawkins J. R., Griffiths B. L., Smith M. J., Foster J. W., Frischauf A. M., Lovell-Badge R., Goodfellow P. N. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature. 1990 Jul 19;346(6281):240–244. doi: 10.1038/346240a0. [DOI] [PubMed] [Google Scholar]
  47. Spolar R. S., Record M. T., Jr Coupling of local folding to site-specific binding of proteins to DNA. Science. 1994 Feb 11;263(5148):777–784. doi: 10.1126/science.8303294. [DOI] [PubMed] [Google Scholar]
  48. Teo S. H., Grasser K. D., Thomas J. O. Differences in the DNA-binding properties of the HMG-box domains of HMG1 and the sex-determining factor SRY. Eur J Biochem. 1995 Jun 15;230(3):943–950. doi: 10.1111/j.1432-1033.1995.tb20640.x. [DOI] [PubMed] [Google Scholar]
  49. Travis A., Amsterdam A., Belanger C., Grosschedl R. LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function [corrected]. Genes Dev. 1991 May;5(5):880–894. doi: 10.1101/gad.5.5.880. [DOI] [PubMed] [Google Scholar]
  50. Ura K., Nightingale K., Wolffe A. P. Differential association of HMG1 and linker histones B4 and H1 with dinucleosomal DNA: structural transitions and transcriptional repression. EMBO J. 1996 Sep 16;15(18):4959–4969. [PMC free article] [PubMed] [Google Scholar]
  51. Weir H. M., Kraulis P. J., Hill C. S., Raine A. R., Laue E. D., Thomas J. O. Structure of the HMG box motif in the B-domain of HMG1. EMBO J. 1993 Apr;12(4):1311–1319. doi: 10.1002/j.1460-2075.1993.tb05776.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wen L., Huang J. K., Johnson B. H., Reeck G. R. A human placental cDNA clone that encodes nonhistone chromosomal protein HMG-1. Nucleic Acids Res. 1989 Feb 11;17(3):1197–1214. doi: 10.1093/nar/17.3.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Werner M. H., Bianchi M. E., Gronenborn A. M., Clore G. M. NMR spectroscopic analysis of the DNA conformation induced by the human testis determining factor SRY. Biochemistry. 1995 Sep 19;34(37):11998–12004. doi: 10.1021/bi00037a042. [DOI] [PubMed] [Google Scholar]
  54. Werner M. H., Huth J. R., Gronenborn A. M., Clore G. M. Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY-DNA complex. Cell. 1995 Jun 2;81(5):705–714. doi: 10.1016/0092-8674(95)90532-4. [DOI] [PubMed] [Google Scholar]
  55. Winkler F. K., Banner D. W., Oefner C., Tsernoglou D., Brown R. S., Heathman S. P., Bryan R. K., Martin P. D., Petratos K., Wilson K. S. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments. EMBO J. 1993 May;12(5):1781–1795. doi: 10.2210/pdb4rve/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Yen Y. M., Wong B., Johnson R. C. Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine. J Biol Chem. 1998 Feb 20;273(8):4424–4435. doi: 10.1074/jbc.273.8.4424. [DOI] [PubMed] [Google Scholar]
  58. Zappavigna V., Falciola L., Helmer-Citterich M., Mavilio F., Bianchi M. E. HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation. EMBO J. 1996 Sep 16;15(18):4981–4991. [PMC free article] [PubMed] [Google Scholar]
  59. Zwilling S., König H., Wirth T. High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors. EMBO J. 1995 Mar 15;14(6):1198–1208. doi: 10.1002/j.1460-2075.1995.tb07103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. van Gent D. C., Hiom K., Paull T. T., Gellert M. Stimulation of V(D)J cleavage by high mobility group proteins. EMBO J. 1997 May 15;16(10):2665–2670. doi: 10.1093/emboj/16.10.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. van Houte L. P., Chuprina V. P., van der Wetering M., Boelens R., Kaptein R., Clevers H. Solution structure of the sequence-specific HMG box of the lymphocyte transcriptional activator Sox-4. J Biol Chem. 1995 Dec 22;270(51):30516–30524. doi: 10.1074/jbc.270.51.30516. [DOI] [PubMed] [Google Scholar]
  62. van de Wetering M., Oosterwegel M., Dooijes D., Clevers H. Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J. 1991 Jan;10(1):123–132. doi: 10.1002/j.1460-2075.1991.tb07928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES