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. 1995 Oct;4(10):1985–1997. doi: 10.1002/pro.5560041004

Water molecules participate in proteinase-inhibitor interactions: crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B.

K Huang 1, W Lu 1, S Anderson 1, M Laskowski Jr 1, M N James 1
PMCID: PMC2142981  PMID: 8535235

Abstract

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water-mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol-1 A-2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site-directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein-complex stability.

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Selected References

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  1. Arnez J. G., Steitz T. A. Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry. 1994 Jun 21;33(24):7560–7567. doi: 10.1021/bi00190a008. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem. 1992 Mar 1;204(2):433–451. doi: 10.1111/j.1432-1033.1992.tb16654.x. [DOI] [PubMed] [Google Scholar]
  3. Bode W., Wei A. Z., Huber R., Meyer E., Travis J., Neumann S. X-ray crystal structure of the complex of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor. EMBO J. 1986 Oct;5(10):2453–2458. doi: 10.1002/j.1460-2075.1986.tb04521.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chothia C. Hydrophobic bonding and accessible surface area in proteins. Nature. 1974 Mar 22;248(446):338–339. doi: 10.1038/248338a0. [DOI] [PubMed] [Google Scholar]
  5. Clackson T., Wells J. A. A hot spot of binding energy in a hormone-receptor interface. Science. 1995 Jan 20;267(5196):383–386. doi: 10.1126/science.7529940. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Eriksson A. E., Baase W. A., Zhang X. J., Heinz D. W., Blaber M., Baldwin E. P., Matthews B. W. Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science. 1992 Jan 10;255(5041):178–183. doi: 10.1126/science.1553543. [DOI] [PubMed] [Google Scholar]
  8. Fujinaga M., Sielecki A. R., Read R. J., Ardelt W., Laskowski M., Jr, James M. N. Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution. J Mol Biol. 1987 May 20;195(2):397–418. doi: 10.1016/0022-2836(87)90659-0. [DOI] [PubMed] [Google Scholar]
  9. Jones T. A. Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. Methods Enzymol. 1985;115:157–171. doi: 10.1016/0076-6879(85)15014-7. [DOI] [PubMed] [Google Scholar]
  10. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  11. Jurásek J., Johnson P., Olafson R. W., Smillie L. B. An improved fractionation system for pronase on CM-sephadex. Can J Biochem. 1971 Nov;49(11):1195–1201. doi: 10.1139/o71-171. [DOI] [PubMed] [Google Scholar]
  12. Kellis J. T., Jr, Nyberg K., Fersht A. R. Energetics of complementary side-chain packing in a protein hydrophobic core. Biochemistry. 1989 May 30;28(11):4914–4922. doi: 10.1021/bi00437a058. [DOI] [PubMed] [Google Scholar]
  13. Kim J. L., Burley S. K. 1.9 A resolution refined structure of TBP recognizing the minor groove of TATAAAAG. Nat Struct Biol. 1994 Sep;1(9):638–653. doi: 10.1038/nsb0994-638. [DOI] [PubMed] [Google Scholar]
  14. Komiyama T., Bigler T. L., Yoshida N., Noda K., Laskowski M., Jr Replacement of P1 Leu18 by Glu18 in the reactive site of turkey ovomucoid third domain converts it into a strong inhibitor of Glu-specific Streptomyces griseus proteinase (GluSGP). J Biol Chem. 1991 Jun 15;266(17):10727–10730. [PubMed] [Google Scholar]
  15. Laskowski M., Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem. 1980;49:593–626. doi: 10.1146/annurev.bi.49.070180.003113. [DOI] [PubMed] [Google Scholar]
  16. Lu W., Zhang W., Molloy S. S., Thomas G., Ryan K., Chiang Y., Anderson S., Laskowski M., Jr Arg15-Lys17-Arg18 turkey ovomucoid third domain inhibits human furin. J Biol Chem. 1993 Jul 15;268(20):14583–14585. [PubMed] [Google Scholar]
  17. Madden D. R., Gorga J. C., Strominger J. L., Wiley D. C. The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC. Cell. 1992 Sep 18;70(6):1035–1048. doi: 10.1016/0092-8674(92)90252-8. [DOI] [PubMed] [Google Scholar]
  18. Musil D., Bode W., Huber R., Laskowski M., Jr, Lin T. Y., Ardelt W. Refined X-ray crystal structures of the reactive site modified ovomucoid inhibitor third domains from silver pheasant (OMSVP3*) and from Japanese quail (OMJPQ3*). J Mol Biol. 1991 Aug 5;220(3):739–755. doi: 10.1016/0022-2836(91)90114-l. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Papamokos E., Weber E., Bode W., Huber R., Empie M. W., Kato I., Laskowski M., Jr Crystallographic refinement of Japanese quail ovomucoid, a Kazal-type inhibitor, and model building studies of complexes with serine proteases. J Mol Biol. 1982 Jul 5;158(3):515–537. doi: 10.1016/0022-2836(82)90212-1. [DOI] [PubMed] [Google Scholar]
  21. Quiocho F. A., Wilson D. K., Vyas N. K. Substrate specificity and affinity of a protein modulated by bound water molecules. Nature. 1989 Aug 3;340(6232):404–407. doi: 10.1038/340404a0. [DOI] [PubMed] [Google Scholar]
  22. Read R. J., Fujinaga M., Sielecki A. R., James M. N. Structure of the complex of Streptomyces griseus protease B and the third domain of the turkey ovomucoid inhibitor at 1.8-A resolution. Biochemistry. 1983 Sep 13;22(19):4420–4433. doi: 10.1021/bi00288a012. [DOI] [PubMed] [Google Scholar]
  23. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  24. Shakked Z., Guzikevich-Guerstein G., Frolow F., Rabinovich D., Joachimiak A., Sigler P. B. Determinants of repressor/operator recognition from the structure of the trp operator binding site. Nature. 1994 Mar 31;368(6470):469–473. doi: 10.1038/368469a0. [DOI] [PubMed] [Google Scholar]
  25. Shortle D., Stites W. E., Meeker A. K. Contributions of the large hydrophobic amino acids to the stability of staphylococcal nuclease. Biochemistry. 1990 Sep 4;29(35):8033–8041. doi: 10.1021/bi00487a007. [DOI] [PubMed] [Google Scholar]
  26. Tronrud D. E. Conjugate-direction minimization: an improved method for the refinement of macromolecules. Acta Crystallogr A. 1992 Nov 1;48(Pt 6):912–916. doi: 10.1107/s0108767392005415. [DOI] [PubMed] [Google Scholar]
  27. Weber E., Papamokos E., Bode W., Huber R., Kato I., Laskowski M., Jr Crystallization, crystal structure analysis and molecular model of the third domain of Japanese quail ovomucoid, a Kazal type inhibitor. J Mol Biol. 1981 Jun 15;149(1):109–123. doi: 10.1016/0022-2836(81)90263-1. [DOI] [PubMed] [Google Scholar]
  28. Wieczorek M., Park S. J., Laskowski M., Jr Covalent hybrids of ovomucoid third domains made from one synthetic and one natural peptide chain. Biochem Biophys Res Commun. 1987 Apr 14;144(1):499–504. doi: 10.1016/s0006-291x(87)80537-5. [DOI] [PubMed] [Google Scholar]

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