Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Mar;3(3):372–390. doi: 10.1002/pro.5560030302

Young Investigator Award Lecture. Structures of larger proteins, protein-ligand and protein-DNA complexes by multidimensional heteronuclear NMR.

G M Clore 1, A M Gronenborn 1
PMCID: PMC2142707  PMID: 8019409

Abstract

The recent development of a whole panoply of multidimensional heteronuclear-edited and -filtered NMR experiments has revolutionized the field of protein structure determination by NMR, making it possible to extend the methodology from the 10-kDa limit of conventional 2-dimensional NMR to systems up to potentially 35-40 kDa. The basic strategy for solving 3-dimensional structures of larger proteins and protein-ligand complexes in solution using 3- and 4-dimensional NMR spectroscopy is summarized, and the power of these methods is illustrated using 3 examples: interleukin-1 beta, the complex of calmodulin with a target peptide, and the specific complex of the transcription factor GATA-1 with its cognate DNA target site.

Full Text

The Full Text of this article is available as a PDF (8.6 MB).

Selected References

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

  1. Babu Y. S., Sack J. S., Greenhough T. J., Bugg C. E., Means A. R., Cook W. J. Three-dimensional structure of calmodulin. Nature. 1985 May 2;315(6014):37–40. doi: 10.1038/315037a0. [DOI] [PubMed] [Google Scholar]
  2. Barbato G., Ikura M., Kay L. E., Pastor R. W., Bax A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry. 1992 Jun 16;31(23):5269–5278. doi: 10.1021/bi00138a005. [DOI] [PubMed] [Google Scholar]
  3. Bennett M. K., Kennedy M. B. Deduced primary structure of the beta subunit of brain type II Ca2+/calmodulin-dependent protein kinase determined by molecular cloning. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1794–1798. doi: 10.1073/pnas.84.7.1794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blumenthal D. K., Krebs E. G. Preparation and properties of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Methods Enzymol. 1987;139:115–126. doi: 10.1016/0076-6879(87)39079-2. [DOI] [PubMed] [Google Scholar]
  5. Braun W. Distance geometry and related methods for protein structure determination from NMR data. Q Rev Biophys. 1987 May;19(3-4):115–157. doi: 10.1017/s0033583500004108. [DOI] [PubMed] [Google Scholar]
  6. Charbonneau H., Kumar S., Novack J. P., Blumenthal D. K., Griffin P. R., Shabanowitz J., Hunt D. F., Beavo J. A., Walsh K. A. Evidence for domain organization within the 61-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain. Biochemistry. 1991 Aug 13;30(32):7931–7940. doi: 10.1021/bi00246a009. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Clore G. M., Driscoll P. C., Wingfield P. T., Gronenborn A. M. Low resolution structure of interleukin-1 beta in solution derived from 1H-15N heteronuclear three-dimensional nuclear magnetic resonance spectroscopy. J Mol Biol. 1990 Aug 20;214(4):811–817. doi: 10.1016/0022-2836(90)90336-k. [DOI] [PubMed] [Google Scholar]
  9. Clore G. M., Gronenborn A. M. Determination of three-dimensional structures of proteins and nucleic acids in solution by nuclear magnetic resonance spectroscopy. Crit Rev Biochem Mol Biol. 1989;24(5):479–564. doi: 10.3109/10409238909086962. [DOI] [PubMed] [Google Scholar]
  10. Clore G. M., Gronenborn A. M. Determination of three-dimensional structures of proteins in solution by nuclear magnetic resonance spectroscopy. Protein Eng. 1987 Aug-Sep;1(4):275–288. doi: 10.1093/protein/1.4.275. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Clore G. M., Wingfield P. T., Gronenborn A. M. High-resolution three-dimensional structure of interleukin 1 beta in solution by three- and four-dimensional nuclear magnetic resonance spectroscopy. Biochemistry. 1991 Mar 5;30(9):2315–2323. doi: 10.1021/bi00223a005. [DOI] [PubMed] [Google Scholar]
  13. Cox J. A., Comte M., Fitton J. E., DeGrado W. F. The interaction of calmodulin with amphiphilic peptides. J Biol Chem. 1985 Feb 25;260(4):2527–2534. [PubMed] [Google Scholar]
  14. Dasgupta M., Honeycutt T., Blumenthal D. K. The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin. J Biol Chem. 1989 Oct 15;264(29):17156–17163. [PubMed] [Google Scholar]
  15. Driscoll P. C., Clore G. M., Marion D., Wingfield P. T., Gronenborn A. M. Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy. Biochemistry. 1990 Apr 10;29(14):3542–3556. doi: 10.1021/bi00466a018. [DOI] [PubMed] [Google Scholar]
  16. Dyson H. J., Gippert G. P., Case D. A., Holmgren A., Wright P. E. Three-dimensional solution structure of the reduced form of Escherichia coli thioredoxin determined by nuclear magnetic resonance spectroscopy. Biochemistry. 1990 May 1;29(17):4129–4136. doi: 10.1021/bi00469a016. [DOI] [PubMed] [Google Scholar]
  17. Eisenberg D., McLachlan A. D. Solvation energy in protein folding and binding. Nature. 1986 Jan 16;319(6050):199–203. doi: 10.1038/319199a0. [DOI] [PubMed] [Google Scholar]
  18. Evans T., Felsenfeld G. The erythroid-specific transcription factor Eryf1: a new finger protein. Cell. 1989 Sep 8;58(5):877–885. doi: 10.1016/0092-8674(89)90940-9. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Finzel B. C., Clancy L. L., Holland D. R., Muchmore S. W., Watenpaugh K. D., Einspahr H. M. Crystal structure of recombinant human interleukin-1 beta at 2.0 A resolution. J Mol Biol. 1989 Oct 20;209(4):779–791. doi: 10.1016/0022-2836(89)90606-2. [DOI] [PubMed] [Google Scholar]
  21. Forman-Kay J. D., Clore G. M., Wingfield P. T., Gronenborn A. M. High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution. Biochemistry. 1991 Mar 12;30(10):2685–2698. doi: 10.1021/bi00224a017. [DOI] [PubMed] [Google Scholar]
  22. Hannon R., Evans T., Felsenfeld G., Gould H. Structure and promoter activity of the gene for the erythroid transcription factor GATA-1. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3004–3008. doi: 10.1073/pnas.88.8.3004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Havel T. F. An evaluation of computational strategies for use in the determination of protein structure from distance constraints obtained by nuclear magnetic resonance. Prog Biophys Mol Biol. 1991;56(1):43–78. doi: 10.1016/0079-6107(91)90007-f. [DOI] [PubMed] [Google Scholar]
  24. Havel T. F., Wüthrich K. An evaluation of the combined use of nuclear magnetic resonance and distance geometry for the determination of protein conformations in solution. J Mol Biol. 1985 Mar 20;182(2):281–294. doi: 10.1016/0022-2836(85)90346-8. [DOI] [PubMed] [Google Scholar]
  25. Heidorn D. B., Seeger P. A., Rokop S. E., Blumenthal D. K., Means A. R., Crespi H., Trewhella J. Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase. Biochemistry. 1989 Aug 8;28(16):6757–6764. doi: 10.1021/bi00442a032. [DOI] [PubMed] [Google Scholar]
  26. Hyberts S. G., Märki W., Wagner G. Stereospecific assignments of side-chain protons and characterization of torsion angles in Eglin c. Eur J Biochem. 1987 May 4;164(3):625–635. doi: 10.1111/j.1432-1033.1987.tb11173.x. [DOI] [PubMed] [Google Scholar]
  27. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  28. Ikura M., Kay L. E., Krinks M., Bax A. Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix. Biochemistry. 1991 Jun 4;30(22):5498–5504. doi: 10.1021/bi00236a024. [DOI] [PubMed] [Google Scholar]
  29. Kataoka M., Head J. F., Persechini A., Kretsinger R. H., Engelman D. M. Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation. Biochemistry. 1991 Feb 5;30(5):1188–1192. doi: 10.1021/bi00219a004. [DOI] [PubMed] [Google Scholar]
  30. Kataoka M., Head J. F., Vorherr T., Krebs J., Carafoli E. Small-angle X-ray scattering study of calmodulin bound to two peptides corresponding to parts of the calmodulin-binding domain of the plasma membrane Ca2+ pump. Biochemistry. 1991 Jun 25;30(25):6247–6251. doi: 10.1021/bi00239a024. [DOI] [PubMed] [Google Scholar]
  31. Kay L. E., Clore G. M., Bax A., Gronenborn A. M. Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution. Science. 1990 Jul 27;249(4967):411–414. doi: 10.1126/science.2377896. [DOI] [PubMed] [Google Scholar]
  32. Kwiatkowski A. P., King M. M. Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation. Biochemistry. 1989 Jun 27;28(13):5380–5385. doi: 10.1021/bi00439a010. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Lukas T. J., Burgess W. H., Prendergast F. G., Lau W., Watterson D. M. Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase. Biochemistry. 1986 Mar 25;25(6):1458–1464. doi: 10.1021/bi00354a041. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Martin D. I., Orkin S. H. Transcriptional activation and DNA binding by the erythroid factor GF-1/NF-E1/Eryf 1. Genes Dev. 1990 Nov;4(11):1886–1898. doi: 10.1101/gad.4.11.1886. [DOI] [PubMed] [Google Scholar]
  37. Nilges M., Clore G. M., Gronenborn A. M. 1H-NMR stereospecific assignments by conformational data-base searches. Biopolymers. 1990 Mar-Apr;29(4-5):813–822. doi: 10.1002/bip.360290415. [DOI] [PubMed] [Google Scholar]
  38. O'Neil K. T., Erickson-Viitanen S., DeGrado W. F. Photolabeling of calmodulin with basic, amphiphilic alpha-helical peptides containing p-benzoylphenylalanine. J Biol Chem. 1989 Aug 25;264(24):14571–14578. [PubMed] [Google Scholar]
  39. Omichinski J. G., Clore G. M., Schaad O., Felsenfeld G., Trainor C., Appella E., Stahl S. J., Gronenborn A. M. NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1. Science. 1993 Jul 23;261(5120):438–446. doi: 10.1126/science.8332909. [DOI] [PubMed] [Google Scholar]
  40. Omichinski J. G., Trainor C., Evans T., Gronenborn A. M., Clore G. M., Felsenfeld G. A small single-"finger" peptide from the erythroid transcription factor GATA-1 binds specifically to DNA as a zinc or iron complex. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1676–1680. doi: 10.1073/pnas.90.5.1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Orkin S. H. GATA-binding transcription factors in hematopoietic cells. Blood. 1992 Aug 1;80(3):575–581. [PubMed] [Google Scholar]
  42. Oschkinat H., Griesinger C., Kraulis P. J., Sørensen O. W., Ernst R. R., Gronenborn A. M., Clore G. M. Three-dimensional NMR spectroscopy of a protein in solution. Nature. 1988 Mar 24;332(6162):374–376. doi: 10.1038/332374a0. [DOI] [PubMed] [Google Scholar]
  43. Pavletich N. P., Pabo C. O. Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers. Science. 1993 Sep 24;261(5129):1701–1707. doi: 10.1126/science.8378770. [DOI] [PubMed] [Google Scholar]
  44. Pavletich N. P., Pabo C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science. 1991 May 10;252(5007):809–817. doi: 10.1126/science.2028256. [DOI] [PubMed] [Google Scholar]
  45. Persechini A., Blumenthal D. K., Jarrett H. W., Klee C. B., Hardy D. O., Kretsinger R. H. The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding. J Biol Chem. 1989 May 15;264(14):8052–8058. [PubMed] [Google Scholar]
  46. Persechini A., Kretsinger R. H. The central helix of calmodulin functions as a flexible tether. J Biol Chem. 1988 Sep 5;263(25):12175–12178. [PubMed] [Google Scholar]
  47. Powers R., Garrett D. S., March C. J., Frieden E. A., Gronenborn A. M., Clore G. M. Three-dimensional solution structure of human interleukin-4 by multidimensional heteronuclear magnetic resonance spectroscopy. Science. 1992 Jun 19;256(5064):1673–1677. doi: 10.1126/science.256.5064.1673. [DOI] [PubMed] [Google Scholar]
  48. Schwabe J. W., Chapman L., Finch J. T., Rhodes D. The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell. 1993 Nov 5;75(3):567–578. doi: 10.1016/0092-8674(93)90390-c. [DOI] [PubMed] [Google Scholar]
  49. Smith L. J., Redfield C., Boyd J., Lawrence G. M., Edwards R. G., Smith R. A., Dobson C. M. Human interleukin 4. The solution structure of a four-helix bundle protein. J Mol Biol. 1992 Apr 20;224(4):899–904. doi: 10.1016/0022-2836(92)90457-u. [DOI] [PubMed] [Google Scholar]
  50. Spera S., Ikura M., Bax A. Measurement of the exchange rates of rapidly exchanging amide protons: application to the study of calmodulin and its complex with a myosin light chain kinase fragment. J Biomol NMR. 1991 Jul;1(2):155–165. doi: 10.1007/BF01877227. [DOI] [PubMed] [Google Scholar]
  51. Thériault Y., Logan T. M., Meadows R., Yu L., Olejniczak E. T., Holzman T. F., Simmer R. L., Fesik S. W. Solution structure of the cyclosporin A/cyclophilin complex by NMR. Nature. 1993 Jan 7;361(6407):88–91. doi: 10.1038/361088a0. [DOI] [PubMed] [Google Scholar]
  52. Trewhella J., Blumenthal D. K., Rokop S. E., Seeger P. A. Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase. Biochemistry. 1990 Oct 9;29(40):9316–9324. doi: 10.1021/bi00492a003. [DOI] [PubMed] [Google Scholar]
  53. Veerapandian B., Gilliland G. L., Raag R., Svensson A. L., Masui Y., Hirai Y., Poulos T. L. Functional implications of interleukin-1 beta based on the three-dimensional structure. Proteins. 1992 Jan;12(1):10–23. doi: 10.1002/prot.340120103. [DOI] [PubMed] [Google Scholar]
  54. Wagner G., Braun W., Havel T. F., Schaumann T., Go N., Wüthrich K. Protein structures in solution by nuclear magnetic resonance and distance geometry. The polypeptide fold of the basic pancreatic trypsin inhibitor determined using two different algorithms, DISGEO and DISMAN. J Mol Biol. 1987 Aug 5;196(3):611–639. doi: 10.1016/0022-2836(87)90037-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES