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. 1998 Sep;7(9):2012–2025. doi: 10.1002/pro.5560070918

Electrostatic coupling to pH-titrating sites as a source of cooperativity in protein-ligand binding.

V Spassov 1, D Bashford 1
PMCID: PMC2144168  PMID: 9761483

Abstract

This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand-binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin D9k, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain.

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Selected References

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  1. Ackers G. K., Shea M. A., Smith F. R. Free energy coupling within macromolecules. The chemical work of ligand binding at the individual sites in co-operative systems. J Mol Biol. 1983 Oct 15;170(1):223–242. doi: 10.1016/s0022-2836(83)80234-4. [DOI] [PubMed] [Google Scholar]
  2. Akke M., Forsén S., Chazin W. J. Solution structure of (Cd2+)1-calbindin D9k reveals details of the stepwise structural changes along the Apo-->(Ca2+)II1-->(Ca2+)I,II2 binding pathway. J Mol Biol. 1995 Sep 8;252(1):102–121. doi: 10.1006/jmbi.1995.0478. [DOI] [PubMed] [Google Scholar]
  3. Bashford D., Gerwert K. Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin. J Mol Biol. 1992 Mar 20;224(2):473–486. doi: 10.1016/0022-2836(92)91009-e. [DOI] [PubMed] [Google Scholar]
  4. Bashford D., Karplus M. pKa's of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry. 1990 Nov 6;29(44):10219–10225. doi: 10.1021/bi00496a010. [DOI] [PubMed] [Google Scholar]
  5. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  6. Beroza P., Fredkin D. R., Okamura M. Y., Feher G. Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5804–5808. doi: 10.1073/pnas.88.13.5804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forsén S., Linse S. Cooperativity: over the Hill. Trends Biochem Sci. 1995 Dec;20(12):495–497. doi: 10.1016/s0968-0004(00)89115-x. [DOI] [PubMed] [Google Scholar]
  8. Gilson M. K. Multiple-site titration and molecular modeling: two rapid methods for computing energies and forces for ionizable groups in proteins. Proteins. 1993 Mar;15(3):266–282. doi: 10.1002/prot.340150305. [DOI] [PubMed] [Google Scholar]
  9. Koshland D. E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966 Jan;5(1):365–385. doi: 10.1021/bi00865a047. [DOI] [PubMed] [Google Scholar]
  10. Linse S., Brodin P., Drakenberg T., Thulin E., Sellers P., Elmdén K., Grundström T., Forsén S. Structure-function relationships in EF-hand Ca2+-binding proteins. Protein engineering and biophysical studies of calbindin D9k. Biochemistry. 1987 Oct 20;26(21):6723–6735. doi: 10.1021/bi00395a023. [DOI] [PubMed] [Google Scholar]
  11. Linse S., Brodin P., Johansson C., Thulin E., Grundström T., Forsén S. The role of protein surface charges in ion binding. Nature. 1988 Oct 13;335(6191):651–652. doi: 10.1038/335651a0. [DOI] [PubMed] [Google Scholar]
  12. Saroff H. A. Ionization of clusters. IV. Cooperativity and allosterism derived from shared clusters in macromolecules. Biopolymers. 1991 Aug;31(9):1037–1047. doi: 10.1002/bip.360310904. [DOI] [PubMed] [Google Scholar]
  13. Saroff H. A. Linked functions and the analysis of individual-site binding data. Anal Biochem. 1993 Jan;208(1):88–95. doi: 10.1006/abio.1993.1012. [DOI] [PubMed] [Google Scholar]
  14. Spassov V. Z., Karshikoff A. D., Ladenstein R. Optimization of the electrostatic interactions in proteins of different functional and folding type. Protein Sci. 1994 Sep;3(9):1556–1569. doi: 10.1002/pro.5560030921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Svensson L. A., Thulin E., Forsén S. Proline cis-trans isomers in calbindin D9k observed by X-ray crystallography. J Mol Biol. 1992 Feb 5;223(3):601–606. doi: 10.1016/0022-2836(92)90976-q. [DOI] [PubMed] [Google Scholar]
  16. Tanford C., Roxby R. Interpretation of protein titration curves. Application to lysozyme. Biochemistry. 1972 May 23;11(11):2192–2198. doi: 10.1021/bi00761a029. [DOI] [PubMed] [Google Scholar]
  17. WYMAN J., Jr LINKED FUNCTIONS AND RECIPROCAL EFFECTS IN HEMOGLOBIN: A SECOND LOOK. Adv Protein Chem. 1964;19:223–286. doi: 10.1016/s0065-3233(08)60190-4. [DOI] [PubMed] [Google Scholar]
  18. Weber G. Energetics of ligand binding to proteins. Adv Protein Chem. 1975;29:1–83. doi: 10.1016/s0065-3233(08)60410-6. [DOI] [PubMed] [Google Scholar]
  19. Yang A. S., Gunner M. R., Sampogna R., Sharp K., Honig B. On the calculation of pKas in proteins. Proteins. 1993 Mar;15(3):252–265. doi: 10.1002/prot.340150304. [DOI] [PubMed] [Google Scholar]
  20. You T. J., Bashford D. Conformation and hydrogen ion titration of proteins: a continuum electrostatic model with conformational flexibility. Biophys J. 1995 Nov;69(5):1721–1733. doi: 10.1016/S0006-3495(95)80042-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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