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. 2001 May;80(5):2082–2092. doi: 10.1016/S0006-3495(01)76182-6

Functional dynamics of the hydrophobic cleft in the N-domain of calmodulin.

D Vigil 1, S C Gallagher 1, J Trewhella 1, A E García 1
PMCID: PMC1301401  PMID: 11325712

Abstract

Molecular dynamics studies of the N-domain (amino acids 1-77; CaM(1-77)) of Ca2+-loaded calmodulin (CaM) show that a solvent exposed hydrophobic cleft in the crystal structure of CaM exhibits transitions from an exposed (open) to a buried (closed) state over a time scale of nanoseconds. As a consequence of burying the hydrophobic cleft, the R(g) of the protein is reduced by 1.5 A. Based on this prediction, x-ray scattering experiments were conducted on this domain over a range of concentrations. Models built from the scattering data show that the R(g) and general shape is consistent with the simulation studies of CaM(1-77). Based on these observations we postulate a model in which the conformation of CaM fluctuates between two different states that expose and bury this hydrophobic cleft. In aqueous solution the closed state dominates the population, while in the presence of peptides, the open state dominates. This inherent flexibility of CaM may be the key to its versatility in recognizing structurally distinct peptide sequences. This model conflicts with the currently accepted hypothesis based on observations in the crystal structure, where upon Ca2+ binding the hydrophobic cleft is exposed to solvent. We postulate that crystal packing forces stabilize the protein conformation toward the open configuration.

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Selected References

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  1. Babu Y. S., Bugg C. E., Cook W. J. Structure of calmodulin refined at 2.2 A resolution. J Mol Biol. 1988 Nov 5;204(1):191–204. doi: 10.1016/0022-2836(88)90608-0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Bentrop D., Bertini I., Cremonini M. A., Forsén S., Luchinat C., Malmendal A. Solution structure of the paramagnetic complex of the N-terminal domain of calmodulin with two Ce3+ ions by 1H NMR. Biochemistry. 1997 Sep 30;36(39):11605–11618. doi: 10.1021/bi971022+. [DOI] [PubMed] [Google Scholar]
  5. Chacón P., Díaz J. F., Morán F., Andreu J. M. Reconstruction of protein form with X-ray solution scattering and a genetic algorithm. J Mol Biol. 2000 Jun 23;299(5):1289–1302. doi: 10.1006/jmbi.2000.3784. [DOI] [PubMed] [Google Scholar]
  6. Chattopadhyaya R., Meador W. E., Means A. R., Quiocho F. A. Calmodulin structure refined at 1.7 A resolution. J Mol Biol. 1992 Dec 20;228(4):1177–1192. doi: 10.1016/0022-2836(92)90324-d. [DOI] [PubMed] [Google Scholar]
  7. Crivici A., Ikura M. Molecular and structural basis of target recognition by calmodulin. Annu Rev Biophys Biomol Struct. 1995;24:85–116. doi: 10.1146/annurev.bb.24.060195.000505. [DOI] [PubMed] [Google Scholar]
  8. Finn B. E., Evenäs J., Drakenberg T., Waltho J. P., Thulin E., Forsén S. Calcium-induced structural changes and domain autonomy in calmodulin. Nat Struct Biol. 1995 Sep;2(9):777–783. doi: 10.1038/nsb0995-777. [DOI] [PubMed] [Google Scholar]
  9. Frishman D., Argos P. Knowledge-based protein secondary structure assignment. Proteins. 1995 Dec;23(4):566–579. doi: 10.1002/prot.340230412. [DOI] [PubMed] [Google Scholar]
  10. Gallagher S. C., Callaghan A. J., Zhao J., Dalton H., Trewhella J. Global conformational changes control the reactivity of methane monooxygenase. Biochemistry. 1999 May 25;38(21):6752–6760. doi: 10.1021/bi982991n. [DOI] [PubMed] [Google Scholar]
  11. García A. E., Hummer G. Water penetration and escape in proteins. Proteins. 2000 Feb 15;38(3):261–272. [PubMed] [Google Scholar]
  12. García AE. Large-amplitude nonlinear motions in proteins. Phys Rev Lett. 1992 Apr 27;68(17):2696–2699. doi: 10.1103/PhysRevLett.68.2696. [DOI] [PubMed] [Google Scholar]
  13. Gellman S. H. On the role of methionine residues in the sequence-independent recognition of nonpolar protein surfaces. Biochemistry. 1991 Jul 9;30(27):6633–6636. doi: 10.1021/bi00241a001. [DOI] [PubMed] [Google Scholar]
  14. Hait W. N., Lazo J. S. Calmodulin: a potential target for cancer chemotherapeutic agents. J Clin Oncol. 1986 Jun;4(6):994–1012. doi: 10.1200/JCO.1986.4.6.994. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Heidorn D. B., Trewhella J. Comparison of the crystal and solution structures of calmodulin and troponin C. Biochemistry. 1988 Feb 9;27(3):909–915. doi: 10.1021/bi00403a011. [DOI] [PubMed] [Google Scholar]
  17. Herzberg O., Moult J., James M. N. A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction. J Biol Chem. 1986 Feb 25;261(6):2638–2644. [PubMed] [Google Scholar]
  18. Humphrey W., Dalke A., Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996 Feb;14(1):33-8, 27-8. doi: 10.1016/0263-7855(96)00018-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. James P., Vorherr T., Carafoli E. Calmodulin-binding domains: just two faced or multi-faceted? Trends Biochem Sci. 1995 Jan;20(1):38–42. doi: 10.1016/s0968-0004(00)88949-5. [DOI] [PubMed] [Google Scholar]
  21. Kawasaki H., Nakayama S., Kretsinger R. H. Classification and evolution of EF-hand proteins. Biometals. 1998 Dec;11(4):277–295. doi: 10.1023/a:1009282307967. [DOI] [PubMed] [Google Scholar]
  22. Krueger J. K., Olah G. A., Rokop S. E., Zhi G., Stull J. T., Trewhella J. Structures of calmodulin and a functional myosin light chain kinase in the activated complex: a neutron scattering study. Biochemistry. 1997 May 20;36(20):6017–6023. doi: 10.1021/bi9702703. [DOI] [PubMed] [Google Scholar]
  23. Krueger J. K., Zhi G., Stull J. T., Trewhella J. Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase. Biochemistry. 1998 Oct 6;37(40):13997–14004. doi: 10.1021/bi981311d. [DOI] [PubMed] [Google Scholar]
  24. Kuboniwa H., Tjandra N., Grzesiek S., Ren H., Klee C. B., Bax A. Solution structure of calcium-free calmodulin. Nat Struct Biol. 1995 Sep;2(9):768–776. doi: 10.1038/nsb0995-768. [DOI] [PubMed] [Google Scholar]
  25. Li M. X., Spyracopoulos L., Sykes B. D. Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C. Biochemistry. 1999 Jun 29;38(26):8289–8298. doi: 10.1021/bi9901679. [DOI] [PubMed] [Google Scholar]
  26. Meador W. E., Means A. R., Quiocho F. A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. 1992 Aug 28;257(5074):1251–1255. doi: 10.1126/science.1519061. [DOI] [PubMed] [Google Scholar]
  27. Mehler E. L., Pascual-Ahuir J. L., Weinstein H. Structural dynamics of calmodulin and troponin C. Protein Eng. 1991 Aug;4(6):625–637. doi: 10.1093/protein/4.6.625. [DOI] [PubMed] [Google Scholar]
  28. Nelson M. R., Chazin W. J. An interaction-based analysis of calcium-induced conformational changes in Ca2+ sensor proteins. Protein Sci. 1998 Feb;7(2):270–282. doi: 10.1002/pro.5560070206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Neil K. T., DeGrado W. F. How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci. 1990 Feb;15(2):59–64. doi: 10.1016/0968-0004(90)90177-d. [DOI] [PubMed] [Google Scholar]
  30. Skelton N. J., Kördel J., Akke M., Forsén S., Chazin W. J. Signal transduction versus buffering activity in Ca(2+)-binding proteins. Nat Struct Biol. 1994 Apr;1(4):239–245. doi: 10.1038/nsb0494-239. [DOI] [PubMed] [Google Scholar]
  31. Svergun D. I. Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J. 1999 Jun;76(6):2879–2886. doi: 10.1016/S0006-3495(99)77443-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vito P., Lacanà E., D'Adamio L. Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer's disease gene ALG-3. Science. 1996 Jan 26;271(5248):521–525. doi: 10.1126/science.271.5248.521. [DOI] [PubMed] [Google Scholar]
  33. Wall M. E., Gallagher S. C., Trewhella J. Large-scale shape changes in proteins and macromolecular complexes. Annu Rev Phys Chem. 2000;51:355–380. doi: 10.1146/annurev.physchem.51.1.355. [DOI] [PubMed] [Google Scholar]
  34. Weinstein H., Mehler E. L. Ca(2+)-binding and structural dynamics in the functions of calmodulin. Annu Rev Physiol. 1994;56:213–236. doi: 10.1146/annurev.ph.56.030194.001241. [DOI] [PubMed] [Google Scholar]
  35. Wilson M. A., Brunger A. T. The 1.0 A crystal structure of Ca(2+)-bound calmodulin: an analysis of disorder and implications for functionally relevant plasticity. J Mol Biol. 2000 Sep 1;301(5):1237–1256. doi: 10.1006/jmbi.2000.4029. [DOI] [PubMed] [Google Scholar]
  36. Wriggers W., Mehler E., Pitici F., Weinstein H., Schulten K. Structure and dynamics of calmodulin in solution. Biophys J. 1998 Apr;74(4):1622–1639. doi: 10.1016/S0006-3495(98)77876-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yap K. L., Ames J. B., Swindells M. B., Ikura M. Diversity of conformational states and changes within the EF-hand protein superfamily. Proteins. 1999 Nov 15;37(3):499–507. doi: 10.1002/(sici)1097-0134(19991115)37:3<499::aid-prot17>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]
  38. Zhang M., Tanaka T., Ikura M. Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nat Struct Biol. 1995 Sep;2(9):758–767. doi: 10.1038/nsb0995-758. [DOI] [PubMed] [Google Scholar]
  39. Zhang M., Yuan T. Molecular mechanisms of calmodulin's functional versatility. Biochem Cell Biol. 1998;76(2-3):313–323. doi: 10.1139/bcb-76-2-3-313. [DOI] [PubMed] [Google Scholar]
  40. Zhao J., Hoye E., Boylan S., Walsh D. A., Trewhella J. Quaternary structures of a catalytic subunit-regulatory subunit dimeric complex and the holoenzyme of the cAMP-dependent protein kinase by neutron contrast variation. J Biol Chem. 1998 Nov 13;273(46):30448–30459. doi: 10.1074/jbc.273.46.30448. [DOI] [PubMed] [Google Scholar]

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