Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Redistribution and loss of side chain entropy upon formation of a calmodulin–peptide complex

Nature Structural Biology volume 7, pages 72–77 (2000)Cite this article

Abstract

The response of the internal dynamics of calcium-saturated calmodulin to the formation of a complex with a peptide model of the calmodulin-binding domain of the smooth muscle myosin light chain kinase has been studied using NMR relaxation methods. The backbone of calmodulin is found to be unaffected by the binding of the domain, whereas the dynamics of side chains are significantly perturbed. The changes in dynamics are interpreted in terms of a heterogeneous partitioning between structure (enthalpy) and dynamics (entropy). These data provide a microscopic view of the residual entropy of a protein in two functional states and suggest extensive enthalpy/entropy exchange during the formation of a protein–protein interface.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Illustration of various motions affecting the obtained generalized order parameters for methyl deuterons.
Figure 2: Methyl symmetry axis order parameters (S2axis) for methyl groups in CaM.
Figure 3: Differences in order parameters (bound - free) in CaM.
Figure 4: Color-coded representation of the side chain dynamics of calmodulin.
Figure 5: Effective correlation times (τe) for methionine methyl groups in a, CaM and b, the CaM–smMLCKp complex.

Similar content being viewed by others

References

  1. Wintrode, P.L. & Privalov, P.L. Energetics of target peptide recognition by calmodulin: a calorimetric study. J. Mol. Biol. 266, 1050–1062 ( 1997).

    Article  CAS  Google Scholar 

  2. Murphy, K.P., Xie, D., Garcia, K.C., Amzel, L.M. & Freire, E. Structural energetics of peptide recognition: angiotensin II/antibody binding. Proteins 15, 113– 120 (1993).

    Article  CAS  Google Scholar 

  3. Lee, K.H., Xie, D., Freire, E. & Amzel, L.M. Estimation of changes in side chain configurational entropy in binding and folding: general methods and application to helix formation. Proteins 20, 68–84 (1994).

    Article  CAS  Google Scholar 

  4. Makhatadze, G.I. & Privalov, P.L. Energetics of protein structure. Adv. Prot. Chem. 47, 307–425 (1995).

    CAS  Google Scholar 

  5. Weber, G. Thermodynamics of the association and the pressure dependence of oligomeric proteins. J. Phys. Chem. 97, 7108– 7115 (1993).

    Article  CAS  Google Scholar 

  6. Karplus, M. & Kushick, J.N. Method for estimating the configurational entropy of macromolecules. Macromolecules 14, 325–332 (1981).

    Article  CAS  Google Scholar 

  7. Doig, A.J. & Sternberg, J.E. Side-chain conformational entropy in protein folding. Protein Sci. 4, 2247 –2251 (1995).

    Article  CAS  Google Scholar 

  8. Rasmussen, B.F., Stock, A.M., Ringe, D. & Petsko, G.A. Crystalline ribonuclease A loses function below the dynamical transition at 220 K. Nature 357, 423–424 ( 1992).

    Article  CAS  Google Scholar 

  9. Feher, V.A. & Cavanagh, J. Millisecond-timescale motions contribute to the function of the bacterial response regulator protein SpoOF. Nature 400, 289–293 ( 1999).

    Article  CAS  Google Scholar 

  10. Stock, A. Relating dynamics to function. Nature 400, 221–222 (1999).

    Article  CAS  Google Scholar 

  11. Akke, M., Brüschweiler, R. & Palmer III, A.G. NMR order parameters and free energy: an analytical approach and its application to cooperative Ca2+ binding by calbindin D9k. J. Am. Chem. Soc. 115, 9832–9833 (1993).

    Article  CAS  Google Scholar 

  12. Li, Z., Raychaudhuri, S. & Wand, A.J. Insights into the local entropy of proteins provided by NMR relaxation. Protein Sci. 5, 2647– 2650 (1996).

    Article  CAS  Google Scholar 

  13. Yang, D. & Kay, L.E. Contributions to conformational entropy arising from bond vector fluctuations measured from NMR-derived order parameters: application to protein folding. J. Mol. Biol. 263, 369–382 (1996).

    Article  CAS  Google Scholar 

  14. Vogel, H.J. Calmodulin: a versatile calcium mediator protein. Biochem. Cell Biol. 72, 357–375 ( 1994).

    Article  CAS  Google Scholar 

  15. Babu, Y.S. et al. Three-dimensional structure of calmodulin. Nature 315, 37–40 ( 1985).

    Article  CAS  Google Scholar 

  16. 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 31, 5269 –5278 (1992).

    Article  CAS  Google Scholar 

  17. Zhang, M., Tanaka, T. & Ikura, M. Calcium-induced conformational transition revealed by the solution structure of apocalmodulin. Nature Struct. Biol. 2, 758–767 (1995).

    Article  CAS  Google Scholar 

  18. Kuboniwa, H. et al. Solution structure of calcium-free calmodulin. Nature Struct. Biol. 2, 768–776 (1995).

    Article  CAS  Google Scholar 

  19. Ikura, M. et al. Solution structure of a calmodulin–target peptide complex by multidimensional NMR. Science 256, 632 –638 (1992).

    Article  CAS  Google Scholar 

  20. Meador, W.E., Means, A.R. & Quiocho, F.A. Target enzyme recognition by calmodulin: 2.4 Å structure of a calmodulin–peptide complex. Science 257, 1251–1256 (1992).

    Article  CAS  Google Scholar 

  21. Meador, W.E., Means, A.R. & Quiocho, F.A. Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science 262, 1718–1721 (1993).

    Article  CAS  Google Scholar 

  22. Osawa, M. et al. A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase. Nature Struct. Biol. 6, 819–824 (1999).

    Article  CAS  Google Scholar 

  23. Muhandiram, D.R., Yamazaki, T., Sykes, B.D. & Kay, L.E. Measurement of 2H T1 and T1ρ relaxation times in uniformly 13C-labeled and fractionally 2H-labeled proteins in solution. J. Am. Chem. Soc. 117, 11536–11544 (1995).

    Article  CAS  Google Scholar 

  24. Lipari, G. & Szabo, A. Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc. 104, 4546–4559 (1982).

    Article  CAS  Google Scholar 

  25. Lee, A.L., Flynn, P.F. & Wand, A.J. Comparison of 2H and 13C NMR relaxation techniques for the study of protein methyl group dynamics in solution. J. Am. Chem. Soc. 121, 2891– 2902 (1999).

    Article  CAS  Google Scholar 

  26. 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 25, 1458 –1464 (1987).

    Article  Google Scholar 

  27. Kemp, B.E., Pearson, R.B., Guerriero, V., Bagchi, I.C. & Means, A.R. The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence. J. Biol. Chem. 262, 2542– 2548 (1987).

    CAS  PubMed  Google Scholar 

  28. Seeholzer, S.H. & Wand, A.J. Structural characterization of the interactions between calmodulin and skeletal muscle myosin light chain kinase: effect of peptide (576–594)G binding on the Ca2+-binding domains. Biochemistry 28, 4011– 4019 (1989).

    Article  CAS  Google Scholar 

  29. Roth, S.M. et al. Structure of the smooth muscle myosin light-chain kinase calmodulin-binding domain peptide bound to calmodulin. Biochemistry 30, 10078–10084 ( 1991).

    Article  CAS  Google Scholar 

  30. Roth, S.M. et al. Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide. Biochemistry 31, 1443–1451 ( 1992).

    Article  CAS  Google Scholar 

  31. Mittermaier, A., Kay, L.E. & Forman-Kay, J.D. Analysis of deuterium relaxation-derived methyl axis order parameters and correlation with local structure. J. Biol. NMR 13, 181–185 ( 1999).

    Article  CAS  Google Scholar 

  32. O'Neil, K.T. & DeGrado, W.F. How calmodulin binds its targets: sequence independent recognition of amphiphilic α-helices. Trends Biochem. Sci. 15, 59–64 (1990).

    Article  CAS  Google Scholar 

  33. Gellman, S.H. On the role of methionine residues in the sequence -independent recognition of nonpolar protein surfaces. Biochemistry 30, 6633–6636 (1991).

    Article  CAS  Google Scholar 

  34. Siivari, K., Zhang, M., Palmer, A.G. & Vogel, H.J. NMR studies of the methionine methyl groups in calmodulin. FEBS Lett. 366, 104–108 (1995).

    Article  CAS  Google Scholar 

  35. Chin, D. & Means, A.R. Methionine to glutamine substitutions in the C-terminal domain of calmodulin impair the activation of three protein kinases. J. Biol. Chem. 271, 30465– 30471 (1996).

    Article  CAS  Google Scholar 

  36. Edwards, R.A., Walsh, M.P., Sutherland, C. & Vogel, H.J. Activation of calcineurin and smooth muscle myosin light chain kinase by Met-to-Leu mutants of calmodulin. Biochem. J. 331, 149–152 (1998).

    Article  CAS  Google Scholar 

  37. Kay, L.E., Muhandiram, D.R., Farrow, N.A., Aubin, Y. & Forman-Kay, J.D. Correlation between dynamics and high affinity binding in an SH2 domain interaction. Biochemistry 35, 361–368 ( 1996).

    Article  CAS  Google Scholar 

  38. Constantine, K.L. et al. Backbone and side chain dynamics of uncomplexed human adipocyte and muscle fatty acid-binding proteins. Biochemistry 37, 7965–7980 ( 1998).

    Article  CAS  Google Scholar 

  39. Gerstein, M., Tsai, J. & Levitt, M. The volume of atoms on the protein surface: calculated from simulation, using Voronoi polyhedra. J. Mol. Biol. 249, 955–966 (1995).

    Article  CAS  Google Scholar 

  40. Jacrot, B., Cusack, S., Dianoux, A.J. & Engelman, D.M. Inelastic neutron scattering analysis of hexokinase dynamics and its modifacation on binding of glucose. Nature 300, 84– 86 (1982).

    Article  CAS  Google Scholar 

  41. Bracken, C., Carr, P.A., Cavanagh, J. & Palmer, A.G. Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA. J. Mol. Biol. 285, 2133–2146 (1999).

    Article  CAS  Google Scholar 

  42. Clackson, T. & Wells, J.A. A hot spot of binding energy in a hormone–receptor interface. Science 267, 383–386 (1995).

    Article  CAS  Google Scholar 

  43. Urbauer, J.L., Short, J.H., Dow, L.K. & Wand, A.J. Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium. Biochemistry 34, 8099–8109 (1995).

    Article  CAS  Google Scholar 

  44. Bax, A., Delaglio, F., Grzesiek, S. & Vuister, G.W. Resonance assignment of methionine methyl groups and χ3 angular information from long-range proton-carbon and carbon-carbon J correlation in a calmodulin–peptide complex. J. Biol. NMR 4, 787–797 (1994).

    Article  CAS  Google Scholar 

  45. Brüschweiler, R., Liao, X. & Wright, P.E. Long-range motional restrictions in a multidomain zinc-finger protein from anisotropic tumbling. Science 268, 886–889 (1995).

    Article  Google Scholar 

  46. Tjandra, N., Feller, S.E., Pastor, R.W. & Bax, A. Rotational diffusion anisotropy of human ubiquitin from 15N NMR relaxation. J. Am. Chem. Soc. 117, 12562–12566 (1995).

    Article  CAS  Google Scholar 

  47. Lee, L.K., Rance, M., Chazin, W.J. & Palmer, A.G. Rotational anisotropy of proteins from simultaneous analysis of 15N and 13CΣϒβα/Σϒβ nuclear spin relaxation. J. Biol. NMR 9, 287–298 (1997).

    Article  CAS  Google Scholar 

  48. Lee, A.L. & Wand, A.J. Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation. J. Biol. NMR 13, 101–112 (1999).

    Article  CAS  Google Scholar 

  49. Brüschweiler, R. & Wright, P.E. NMR order parameters of biomolecules: a new analytical representation and application to the Gaussian axial fluctuation model. J. Am. Chem. Soc. 116, 8426–8427 (1994).

    Article  Google Scholar 

  50. Koradi, R., Billeter, M. & Wüthrich, K. A program for display and analysis of macromolecular structures. J. Mol. Graphics 14, 51– 55 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Sharp and J. Kranz for helpful discussions. This work was supported by a grant and a postdoctoral fellowship from the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, The Johnson Research Foundation, School of Medicine, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Andrew L. Lee, Sandra A. Kinnear & A. Joshua Wand

Authors
  1. Andrew L. Lee
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Sandra A. Kinnear
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. A. Joshua Wand
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to A. Joshua Wand.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, A., Kinnear, S. & Wand, A. Redistribution and loss of side chain entropy upon formation of a calmodulin–peptide complex. Nat Struct Mol Biol 7, 72–77 (2000). https://doi.org/10.1038/71280

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/71280

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing