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 Feb;9(2):252–264. doi: 10.1110/ps.9.2.252

Structure-based thermodynamic analysis of the dissociation of protein phosphatase-1 catalytic subunit and microcystin-LR docked complexes.

P Lavigne 1, J R Bagu 1, R Boyko 1, L Willard 1, C F Holmes 1, B D Sykes 1
PMCID: PMC2144542  PMID: 10716177

Abstract

The relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure-based thermodynamic approach to study the dissociation of the complex between the toxin microcystin-LR (MLR) and the catalytic domain of protein phosphatase-1 (PP-1c) for which the crystal structure of the complex is known. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy) for the dissociation of the complex from its X-ray structure and found the calculated dissociation constant (4.0 x 10(-11)) to be in excellent agreement with the reported inhibitory constant (3.9 x 10(-11)). We have also calculated the thermodynamic parameters for the dissociation of 47 PP-1c:MLR complexes generated by docking an ensemble of NMR solution structures of MLR onto the crystal structure of PP-1c. In general, we observe that the lower the root-mean-square deviation (RMSD) of the docked complex (compared to the X-ray complex) the closer its free energy of dissociation (deltaGd(o)) is to that calculated from the X-ray complex. On the other hand, we note a significant scatter between the deltaGd(o) and the RMSD of the docked complexes. We have identified a group of seven docked complexes with deltaGd(o) values very close to the one calculated from the X-ray complex but with significantly dissimilar structures. The analysis of the corresponding enthalpy and entropy of dissociation shows a compensation effect suggesting that MLR molecules with significant structural variability can bind PP-1c and that substantial conformational flexibility in the PP-1c:MLR complex may exist in solution.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

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

  1. Abagyan R., Totrov M. Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. J Mol Biol. 1994 Jan 21;235(3):983–1002. doi: 10.1006/jmbi.1994.1052. [DOI] [PubMed] [Google Scholar]
  2. Bagu J. R., Sykes B. D., Craig M. M., Holmes C. F. A molecular basis for different interactions of marine toxins with protein phosphatase-1. Molecular models for bound motuporin, microcystins, okadaic acid, and calyculin A. J Biol Chem. 1997 Feb 21;272(8):5087–5097. doi: 10.1074/jbc.272.8.5087. [DOI] [PubMed] [Google Scholar]
  3. Bagu J. R., Sönnichsen F. D., Williams D., Andersen R. J., Sykes B. D., Holmes C. F. Comparison of the solution structures of microcystin-LR and motuporin. Nat Struct Biol. 1995 Feb;2(2):114–116. doi: 10.1038/nsb0295-114. [DOI] [PubMed] [Google Scholar]
  4. Baker B. M., Murphy K. P. Dissecting the energetics of a protein-protein interaction: the binding of ovomucoid third domain to elastase. J Mol Biol. 1997 May 2;268(2):557–569. doi: 10.1006/jmbi.1997.0977. [DOI] [PubMed] [Google Scholar]
  5. Baker B. M., Murphy K. P. Prediction of binding energetics from structure using empirical parameterization. Methods Enzymol. 1998;295:294–315. doi: 10.1016/s0076-6879(98)95045-5. [DOI] [PubMed] [Google Scholar]
  6. Baldwin R. L. Temperature dependence of the hydrophobic interaction in protein folding. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8069–8072. doi: 10.1073/pnas.83.21.8069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bardi J. S., Luque I., Freire E. Structure-based thermodynamic analysis of HIV-1 protease inhibitors. Biochemistry. 1997 Jun 3;36(22):6588–6596. doi: 10.1021/bi9701742. [DOI] [PubMed] [Google Scholar]
  8. Barford D. Protein phosphatases. Curr Opin Struct Biol. 1995 Dec;5(6):728–734. doi: 10.1016/0959-440x(95)80004-2. [DOI] [PubMed] [Google Scholar]
  9. Brady G. P., Sharp K. A. Entropy in protein folding and in protein-protein interactions. Curr Opin Struct Biol. 1997 Apr;7(2):215–221. doi: 10.1016/s0959-440x(97)80028-0. [DOI] [PubMed] [Google Scholar]
  10. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  11. Cooper A. Thermodynamic fluctuations in protein molecules. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2740–2741. doi: 10.1073/pnas.73.8.2740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Craig M., McCready T. L., Luu H. A., Smillie M. A., Dubord P., Holmes C. F. Identification and characterization of hydrophobic microcystins in Canadian freshwater cyanobacteria. Toxicon. 1993 Dec;31(12):1541–1549. doi: 10.1016/0041-0101(93)90338-j. [DOI] [PubMed] [Google Scholar]
  13. Cummings M. D., Hart T. N., Read R. J. Atomic solvation parameters in the analysis of protein-protein docking results. Protein Sci. 1995 Oct;4(10):2087–2099. doi: 10.1002/pro.5560041014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. D'Aquino J. A., Gómez J., Hilser V. J., Lee K. H., Amzel L. M., Freire E. The magnitude of the backbone conformational entropy change in protein folding. Proteins. 1996 Jun;25(2):143–156. doi: 10.1002/(SICI)1097-0134(199606)25:2<143::AID-PROT1>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  15. Dauber-Osguthorpe P., Roberts V. A., Osguthorpe D. J., Wolff J., Genest M., Hagler A. T. Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins. 1988;4(1):31–47. doi: 10.1002/prot.340040106. [DOI] [PubMed] [Google Scholar]
  16. Eftink M. R., Anusiem A. C., Biltonen R. L. Enthalpy-entropy compensation and heat capacity changes for protein-ligand interactions: general thermodynamic models and data for the binding of nucleotides to ribonuclease A. Biochemistry. 1983 Aug 2;22(16):3884–3896. doi: 10.1021/bi00285a025. [DOI] [PubMed] [Google Scholar]
  17. Egloff M. P., Cohen P. T., Reinemer P., Barford D. Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. J Mol Biol. 1995 Dec 15;254(5):942–959. doi: 10.1006/jmbi.1995.0667. [DOI] [PubMed] [Google Scholar]
  18. Frauenfelder H., Parak F., Young R. D. Conformational substates in proteins. Annu Rev Biophys Biophys Chem. 1988;17:451–479. doi: 10.1146/annurev.bb.17.060188.002315. [DOI] [PubMed] [Google Scholar]
  19. Gilson M. K., Given J. A., Bush B. L., McCammon J. A. The statistical-thermodynamic basis for computation of binding affinities: a critical review. Biophys J. 1997 Mar;72(3):1047–1069. doi: 10.1016/S0006-3495(97)78756-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldberg J., Huang H. B., Kwon Y. G., Greengard P., Nairn A. C., Kuriyan J. Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature. 1995 Aug 31;376(6543):745–753. doi: 10.1038/376745a0. [DOI] [PubMed] [Google Scholar]
  21. Gómez J., Freire E. Thermodynamic mapping of the inhibitor site of the aspartic protease endothiapepsin. J Mol Biol. 1995 Sep 22;252(3):337–350. doi: 10.1006/jmbi.1995.0501. [DOI] [PubMed] [Google Scholar]
  22. Gómez J., Hilser V. J., Xie D., Freire E. The heat capacity of proteins. Proteins. 1995 Aug;22(4):404–412. doi: 10.1002/prot.340220410. [DOI] [PubMed] [Google Scholar]
  23. Hawkes R., Grutter M. G., Schellman J. Thermodynamic stability and point mutations of bacteriophage T4 lysozyme. J Mol Biol. 1984 May 15;175(2):195–212. doi: 10.1016/0022-2836(84)90474-1. [DOI] [PubMed] [Google Scholar]
  24. Hilser V. J., Gómez J., Freire E. The enthalpy change in protein folding and binding: refinement of parameters for structure-based calculations. Proteins. 1996 Oct;26(2):123–133. doi: 10.1002/(SICI)1097-0134(199610)26:2<123::AID-PROT2>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  25. Holtzer A. The "cratic correction" and related fallacies. Biopolymers. 1995 Jun;35(6):595–602. doi: 10.1002/bip.360350605. [DOI] [PubMed] [Google Scholar]
  26. Huang H. B., Horiuchi A., Goldberg J., Greengard P., Nairn A. C. Site-directed mutagenesis of amino acid residues of protein phosphatase 1 involved in catalysis and inhibitor binding. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3530–3535. doi: 10.1073/pnas.94.8.3530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jackson R. M., Sternberg M. J. A continuum model for protein-protein interactions: application to the docking problem. J Mol Biol. 1995 Jul 7;250(2):258–275. doi: 10.1006/jmbi.1995.0375. [DOI] [PubMed] [Google Scholar]
  28. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  29. 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. 1994 Sep;20(1):68–84. doi: 10.1002/prot.340200108. [DOI] [PubMed] [Google Scholar]
  30. Lumry R., Rajender S. Enthalpy-entropy compensation phenomena in water solutions of proteins and small molecules: a ubiquitous property of water. Biopolymers. 1970;9(10):1125–1227. doi: 10.1002/bip.1970.360091002. [DOI] [PubMed] [Google Scholar]
  31. MacKintosh R. W., Dalby K. N., Campbell D. G., Cohen P. T., Cohen P., MacKintosh C. The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1. FEBS Lett. 1995 Sep 11;371(3):236–240. doi: 10.1016/0014-5793(95)00888-g. [DOI] [PubMed] [Google Scholar]
  32. Makhatadze G. I., Privalov P. L. Energetics of protein structure. Adv Protein Chem. 1995;47:307–425. doi: 10.1016/s0065-3233(08)60548-3. [DOI] [PubMed] [Google Scholar]
  33. Miller S., Janin J., Lesk A. M., Chothia C. Interior and surface of monomeric proteins. J Mol Biol. 1987 Aug 5;196(3):641–656. doi: 10.1016/0022-2836(87)90038-6. [DOI] [PubMed] [Google Scholar]
  34. Moult J. Comparison of database potentials and molecular mechanics force fields. Curr Opin Struct Biol. 1997 Apr;7(2):194–199. doi: 10.1016/s0959-440x(97)80025-5. [DOI] [PubMed] [Google Scholar]
  35. Murphy K. P., Freire E. Thermodynamics of structural stability and cooperative folding behavior in proteins. Adv Protein Chem. 1992;43:313–361. doi: 10.1016/s0065-3233(08)60556-2. [DOI] [PubMed] [Google Scholar]
  36. Murphy K. P., Xie D., Thompson K. S., Amzel L. M., Freire E. Entropy in biological binding processes: estimation of translational entropy loss. Proteins. 1994 Jan;18(1):63–67. doi: 10.1002/prot.340180108. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Nishiwaki-Matsushima R., Nishiwaki S., Ohta T., Yoshizawa S., Suganuma M., Harada K., Watanabe M. F., Fujiki H. Structure-function relationships of microcystins, liver tumor promoters, in interaction with protein phosphatase. Jpn J Cancer Res. 1991 Sep;82(9):993–996. doi: 10.1111/j.1349-7006.1991.tb01933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Novotny J., Bruccoleri R. E., Davis M., Sharp K. A. Empirical free energy calculations: a blind test and further improvements to the method. J Mol Biol. 1997 May 2;268(2):401–411. doi: 10.1006/jmbi.1997.0961. [DOI] [PubMed] [Google Scholar]
  40. Ohta T., Nishiwaki R., Yatsunami J., Komori A., Suganuma M., Fujiki H. Hyperphosphorylation of cytokeratins 8 and 18 by microcystin-LR, a new liver tumor promoter, in primary cultured rat hepatocytes. Carcinogenesis. 1992 Dec;13(12):2443–2447. doi: 10.1093/carcin/13.12.2443. [DOI] [PubMed] [Google Scholar]
  41. Pickett S. D., Sternberg M. J. Empirical scale of side-chain conformational entropy in protein folding. J Mol Biol. 1993 Jun 5;231(3):825–839. doi: 10.1006/jmbi.1993.1329. [DOI] [PubMed] [Google Scholar]
  42. Richmond T. J. Solvent accessible surface area and excluded volume in proteins. Analytical equations for overlapping spheres and implications for the hydrophobic effect. J Mol Biol. 1984 Sep 5;178(1):63–89. doi: 10.1016/0022-2836(84)90231-6. [DOI] [PubMed] [Google Scholar]
  43. Runnegar M., Berndt N., Kong S. M., Lee E. Y., Zhang L. In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2A. Biochem Biophys Res Commun. 1995 Nov 2;216(1):162–169. doi: 10.1006/bbrc.1995.2605. [DOI] [PubMed] [Google Scholar]
  44. Shenolikar S. Protein serine/threonine phosphatases--new avenues for cell regulation. Annu Rev Cell Biol. 1994;10:55–86. doi: 10.1146/annurev.cb.10.110194.000415. [DOI] [PubMed] [Google Scholar]
  45. Shortle D., Meeker A. K., Freire E. Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction. Biochemistry. 1988 Jun 28;27(13):4761–4768. doi: 10.1021/bi00413a027. [DOI] [PubMed] [Google Scholar]
  46. Stotts R. R., Namikoshi M., Haschek W. M., Rinehart K. L., Carmichael W. W., Dahlem A. M., Beasley V. R. Structural modifications imparting reduced toxicity in microcystins from Microcystis spp. Toxicon. 1993 Jun;31(6):783–789. doi: 10.1016/0041-0101(93)90384-u. [DOI] [PubMed] [Google Scholar]
  47. Takai A., Sasaki K., Nagai H., Mieskes G., Isobe M., Isono K., Yasumoto T. Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein. Biochem J. 1995 Mar 15;306(Pt 3):657–665. doi: 10.1042/bj3060657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tamura A., Privalov P. L. The entropy cost of protein association. J Mol Biol. 1997 Nov 14;273(5):1048–1060. doi: 10.1006/jmbi.1997.1368. [DOI] [PubMed] [Google Scholar]
  49. Vajda S., Sippl M., Novotny J. Empirical potentials and functions for protein folding and binding. Curr Opin Struct Biol. 1997 Apr;7(2):222–228. doi: 10.1016/s0959-440x(97)80029-2. [DOI] [PubMed] [Google Scholar]
  50. Weng Z., Vajda S., Delisi C. Prediction of protein complexes using empirical free energy functions. Protein Sci. 1996 Apr;5(4):614–626. doi: 10.1002/pro.5560050406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Xie D., Freire E. Molecular basis of cooperativity in protein folding. V. Thermodynamic and structural conditions for the stabilization of compact denatured states. Proteins. 1994 Aug;19(4):291–301. doi: 10.1002/prot.340190404. [DOI] [PubMed] [Google Scholar]

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

RESOURCES