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
. 1998 Oct;7(10):2054–2064. doi: 10.1002/pro.5560071002

Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity.

P C Sanschagrin 1, L A Kuhn 1
PMCID: PMC2143843  PMID: 9792092

Abstract

Cluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water molecules, and also avoids reliance on any one structure as a reference. Water sites were clustered for ten superimposed thrombin structures, three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water molecules from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concentrated in buried regions including the solvent channel surrounding the Na+ site in thrombin, which is associated with ligand selectivity. Cluster analysis also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addition to facilitating the analysis of water sites from multiple structures, cluster analysis provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.

Full Text

The Full Text of this article is available as a PDF (4.3 MB).

Selected References

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

  1. Badger J. Multiple hydration layers in cubic insulin crystals. Biophys J. 1993 Oct;65(4):1656–1659. doi: 10.1016/S0006-3495(93)81220-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Blow D. M., Birktoft J. J., Hartley B. S. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature. 1969 Jan 25;221(5178):337–340. doi: 10.1038/221337a0. [DOI] [PubMed] [Google Scholar]
  4. Braden B. C., Fields B. A., Poljak R. J. Conservation of water molecules in an antibody-antigen interaction. J Mol Recognit. 1995 Sep-Oct;8(5):317–325. doi: 10.1002/jmr.300080505. [DOI] [PubMed] [Google Scholar]
  5. Brunne R. M., Liepinsh E., Otting G., Wüthrich K., van Gunsteren W. F. Hydration of proteins. A comparison of experimental residence times of water molecules solvating the bovine pancreatic trypsin inhibitor with theoretical model calculations. J Mol Biol. 1993 Jun 20;231(4):1040–1048. doi: 10.1006/jmbi.1993.1350. [DOI] [PubMed] [Google Scholar]
  6. Burling F. T., Weis W. I., Flaherty K. M., Brünger A. T. Direct observation of protein solvation and discrete disorder with experimental crystallographic phases. Science. 1996 Jan 5;271(5245):72–77. doi: 10.1126/science.271.5245.72. [DOI] [PubMed] [Google Scholar]
  7. Connolly M. L. The molecular surface package. J Mol Graph. 1993 Jun;11(2):139–141. doi: 10.1016/0263-7855(93)87010-3. [DOI] [PubMed] [Google Scholar]
  8. Craig L., Sanschagrin P. C., Rozek A., Lackie S., Kuhn L. A., Scott J. K. The role of structure in antibody cross-reactivity between peptides and folded proteins. J Mol Biol. 1998 Aug 7;281(1):183–201. doi: 10.1006/jmbi.1998.1907. [DOI] [PubMed] [Google Scholar]
  9. Dang Q. D., Di Cera E. Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10653–10656. doi: 10.1073/pnas.93.20.10653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Denisov V. P., Peters J., Hörlein H. D., Halle B. Using buried water molecules to explore the energy landscape of proteins. Nat Struct Biol. 1996 Jun;3(6):505–509. doi: 10.1038/nsb0696-505. [DOI] [PubMed] [Google Scholar]
  11. Di Cera E., Guinto E. R., Vindigni A., Dang Q. D., Ayala Y. M., Wuyi M., Tulinsky A. The Na+ binding site of thrombin. J Biol Chem. 1995 Sep 22;270(38):22089–22092. doi: 10.1074/jbc.270.38.22089. [DOI] [PubMed] [Google Scholar]
  12. Earnest T., Fauman E., Craik C. S., Stroud R. 1.59 A structure of trypsin at 120 K: comparison of low temperature and room temperature structures. Proteins. 1991;10(3):171–187. doi: 10.1002/prot.340100303. [DOI] [PubMed] [Google Scholar]
  13. Edsall J. T., McKenzie H. A. Water and proteins. II. The location and dynamics of water in protein systems and its relation to their stability and properties. Adv Biophys. 1983;16:53–183. doi: 10.1016/0065-227x(83)90008-4. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Esmon C. T. The protein C anticoagulant pathway. Arterioscler Thromb. 1992 Feb;12(2):135–145. doi: 10.1161/01.atv.12.2.135. [DOI] [PubMed] [Google Scholar]
  16. Finer-Moore J. S., Kossiakoff A. A., Hurley J. H., Earnest T., Stroud R. M. Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction. Proteins. 1992 Mar;12(3):203–222. doi: 10.1002/prot.340120302. [DOI] [PubMed] [Google Scholar]
  17. Furie B., Furie B. C. The molecular basis of blood coagulation. Cell. 1988 May 20;53(4):505–518. doi: 10.1016/0092-8674(88)90567-3. [DOI] [PubMed] [Google Scholar]
  18. Joachimiak A., Haran T. E., Sigler P. B. Mutagenesis supports water mediated recognition in the trp repressor-operator system. EMBO J. 1994 Jan 15;13(2):367–372. doi: 10.1002/j.1460-2075.1994.tb06270.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kossiakoff A. A., Sintchak M. D., Shpungin J., Presta L. G. Analysis of solvent structure in proteins using neutron D2O-H2O solvent maps: pattern of primary and secondary hydration of trypsin. Proteins. 1992 Mar;12(3):223–236. doi: 10.1002/prot.340120303. [DOI] [PubMed] [Google Scholar]
  20. Kuhn L. A., Siani M. A., Pique M. E., Fisher C. L., Getzoff E. D., Tainer J. A. The interdependence of protein surface topography and bound water molecules revealed by surface accessibility and fractal density measures. J Mol Biol. 1992 Nov 5;228(1):13–22. doi: 10.1016/0022-2836(92)90487-5. [DOI] [PubMed] [Google Scholar]
  21. Kuhn L. A., Swanson C. A., Pique M. E., Tainer J. A., Getzoff E. D. Atomic and residue hydrophilicity in the context of folded protein structures. Proteins. 1995 Dec;23(4):536–547. doi: 10.1002/prot.340230408. [DOI] [PubMed] [Google Scholar]
  22. Kuntz I. D., Jr, Kauzmann W. Hydration of proteins and polypeptides. Adv Protein Chem. 1974;28:239–345. doi: 10.1016/s0065-3233(08)60232-6. [DOI] [PubMed] [Google Scholar]
  23. Ladbury J. E. Just add water! The effect of water on the specificity of protein-ligand binding sites and its potential application to drug design. Chem Biol. 1996 Dec;3(12):973–980. doi: 10.1016/s1074-5521(96)90164-7. [DOI] [PubMed] [Google Scholar]
  24. Lam P. Y., Jadhav P. K., Eyermann C. J., Hodge C. N., Ru Y., Bacheler L. T., Meek J. L., Otto M. J., Rayner M. M., Wong Y. N. Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors. Science. 1994 Jan 21;263(5145):380–384. doi: 10.1126/science.8278812. [DOI] [PubMed] [Google Scholar]
  25. Levitt M., Park B. H. Water: now you see it, now you don't. Structure. 1993 Dec 15;1(4):223–226. doi: 10.1016/0969-2126(93)90011-5. [DOI] [PubMed] [Google Scholar]
  26. Meyer E. Internal water molecules and H-bonding in biological macromolecules: a review of structural features with functional implications. Protein Sci. 1992 Dec;1(12):1543–1562. doi: 10.1002/pro.5560011203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mikol V., Papageorgiou C., Borer X. The role of water molecules in the structure-based design of (5-hydroxynorvaline)-2-cyclosporin: synthesis, biological activity, and crystallographic analysis with cyclophilin A. J Med Chem. 1995 Aug 18;38(17):3361–3367. doi: 10.1021/jm00017a020. [DOI] [PubMed] [Google Scholar]
  28. Otting G., Liepinsh E., Wüthrich K. Protein hydration in aqueous solution. Science. 1991 Nov 15;254(5034):974–980. doi: 10.1126/science.1948083. [DOI] [PubMed] [Google Scholar]
  29. Otwinowski Z., Schevitz R. W., Zhang R. G., Lawson C. L., Joachimiak A., Marmorstein R. Q., Luisi B. F., Sigler P. B. Crystal structure of trp repressor/operator complex at atomic resolution. Nature. 1988 Sep 22;335(6188):321–329. doi: 10.1038/335321a0. [DOI] [PubMed] [Google Scholar]
  30. Perona J. J., Craik C. S., Fletterick R. J. Locating the catalytic water molecule in serine proteases. Science. 1993 Jul 30;261(5121):620–622. doi: 10.1126/science.8342029. [DOI] [PubMed] [Google Scholar]
  31. Rashin A. A., Iofin M., Honig B. Internal cavities and buried waters in globular proteins. Biochemistry. 1986 Jun 17;25(12):3619–3625. doi: 10.1021/bi00360a021. [DOI] [PubMed] [Google Scholar]
  32. Raymer M. L., Sanschagrin P. C., Punch W. F., Venkataraman S., Goodman E. D., Kuhn L. A. Predicting conserved water-mediated and polar ligand interactions in proteins using a K-nearest-neighbors genetic algorithm. J Mol Biol. 1997 Jan 31;265(4):445–464. doi: 10.1006/jmbi.1996.0746. [DOI] [PubMed] [Google Scholar]
  33. Ringe D. What makes a binding site a binding site? Curr Opin Struct Biol. 1995 Dec;5(6):825–829. doi: 10.1016/0959-440x(95)80017-4. [DOI] [PubMed] [Google Scholar]
  34. Schiffer C. A., Huber R., Wüthrich K., van Gunsteren W. F. Simultaneous refinement of the structure of BPTI against NMR data measured in solution and X-ray diffraction data measured in single crystals. J Mol Biol. 1994 Aug 26;241(4):588–599. doi: 10.1006/jmbi.1994.1533. [DOI] [PubMed] [Google Scholar]
  35. Singer P. T., Smalås A., Carty R. P., Mangel W. F., Sweet R. M. The hydrolytic water molecule in trypsin, revealed by time-resolved Laue crystallography. Science. 1993 Jan 29;259(5095):669–673. doi: 10.1126/science.8430314. [DOI] [PubMed] [Google Scholar]
  36. Sreenivasan U., Axelsen P. H. Buried water in homologous serine proteases. Biochemistry. 1992 Dec 29;31(51):12785–12791. doi: 10.1021/bi00166a011. [DOI] [PubMed] [Google Scholar]
  37. Wang H., Ben-Naim A. A possible involvement of solvent-induced interactions in drug design. J Med Chem. 1996 Mar 29;39(7):1531–1539. doi: 10.1021/jm950430d. [DOI] [PubMed] [Google Scholar]
  38. Williams M. A., Goodfellow J. M., Thornton J. M. Buried waters and internal cavities in monomeric proteins. Protein Sci. 1994 Aug;3(8):1224–1235. doi: 10.1002/pro.5560030808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wilson I. A., Fremont D. H. Structural analysis of MHC class I molecules with bound peptide antigens. Semin Immunol. 1993 Apr;5(2):75–80. doi: 10.1006/smim.1993.1011. [DOI] [PubMed] [Google Scholar]
  40. Zhang E., Tulinsky A. The molecular environment of the Na+ binding site of thrombin. Biophys Chem. 1997 Jan 31;63(2-3):185–200. doi: 10.1016/s0301-4622(96)02227-2. [DOI] [PubMed] [Google Scholar]
  41. Zhang X. J., Matthews B. W. Conservation of solvent-binding sites in 10 crystal forms of T4 lysozyme. Protein Sci. 1994 Jul;3(7):1031–1039. doi: 10.1002/pro.5560030705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van Gunsteren W. F., Berendsen H. J., Hermans J., Hol W. G., Postma J. P. Computer simulation of the dynamics of hydrated protein crystals and its comparison with x-ray data. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4315–4319. doi: 10.1073/pnas.80.14.4315. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES