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. 1995 Sep;4(9):1683–1695. doi: 10.1002/pro.5560040904

Synthesis, activity, and preliminary structure of the fourth EGF-like domain of thrombomodulin.

D P Meininger 1, M J Hunter 1, E A Komives 1
PMCID: PMC2143218  PMID: 8528067

Abstract

The fourth EGF-like domain of thrombomodulin (TM4), residues E346-F389 in the TM sequence, has been synthesized. Refolding of the synthetic product under redox conditions gave a single major product. The disulfide bonding pattern of the folded, oxidized domain was (1-3, 2-4, 5-6), which is the same as that found in EGF protein. TM4 was tested for TM anticoagulant activity because deletion and substitution mutagenesis experiments have shown that the fourth EGF-like domain of TM is essential for TM cofactor activity. TM4 showed no TM-like activity in two assay systems, both for inhibition of fibrin clot formation, and for cofactor activity in thrombin activation of protein C. A preliminary structure of TM4 was determined by 2D 1H NMR from 519 NOE-derived distance constraints. Distance geometry calculations yielded a single convergent structure. The structure resembles the structure of EGF and other known EGF-like domains but has some key differences. The central two-stranded beta-sheet is conserved despite the differences in the number of amino acids in the loops. The C-terminal loop formed by the disulfide bond between C372 and C386 in TM4 is five amino acids longer than the analogous loop between C33 and C42 of EGF protein. This loop appears to have a different fold in TM4 than in EGF protein. The loop forms the two outside strands of a broken, irregular tri-stranded beta-sheet, and amino acids H384-F389 lie between the two strands forming the middle strand of the sheet. Thus, although the C-terminus of EGF protein forms one of the outside strands of a tri-stranded antiparallel sheet, the C-terminus of TM4 forms the inside strand of an irregular tri-stranded parallel-anti-parallel sheet. The residues D349, E357, and E374, which were shown to be critical for cofactor activity by alanine scanning mutagenesis, all lie in a patch near the C-terminal loop, and are solvent accessible. The other critical residues, Y358 and F376, are largely buried and appear to play essential structural rather than functional roles.

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Selected References

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  1. Baron M., Norman D. G., Harvey T. S., Handford P. A., Mayhew M., Tse A. G., Brownlee G. G., Campbell I. D. The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha. Protein Sci. 1992 Jan;1(1):81–90. doi: 10.1002/pro.5560010109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Braun W., Go N. Calculation of protein conformations by proton-proton distance constraints. A new efficient algorithm. J Mol Biol. 1985 Dec 5;186(3):611–626. doi: 10.1016/0022-2836(85)90134-2. [DOI] [PubMed] [Google Scholar]
  3. Clubb R. T., Ferguson S. B., Walsh C. T., Wagner G. Three-dimensional solution structure of Escherichia coli periplasmic cyclophilin. Biochemistry. 1994 Mar 15;33(10):2761–2772. doi: 10.1021/bi00176a004. [DOI] [PubMed] [Google Scholar]
  4. Diamond R. On the multiple simultaneous superposition of molecular structures by rigid body transformations. Protein Sci. 1992 Oct;1(10):1279–1287. doi: 10.1002/pro.5560011006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Esmon N. L. Thrombomodulin. Prog Hemost Thromb. 1989;9:29–55. [PubMed] [Google Scholar]
  6. Glaser C. B., Morser J., Clarke J. H., Blasko E., McLean K., Kuhn I., Chang R. J., Lin J. H., Vilander L., Andrews W. H. Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation. J Clin Invest. 1992 Dec;90(6):2565–2573. doi: 10.1172/JCI116151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graves B. J., Crowther R. L., Chandran C., Rumberger J. M., Li S., Huang K. S., Presky D. H., Familletti P. C., Wolitzky B. A., Burns D. K. Insight into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains. Nature. 1994 Feb 10;367(6463):532–538. doi: 10.1038/367532a0. [DOI] [PubMed] [Google Scholar]
  8. Gray W. R. Disulfide structures of highly bridged peptides: a new strategy for analysis. Protein Sci. 1993 Oct;2(10):1732–1748. doi: 10.1002/pro.5560021017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Güntert P., Braun W., Wüthrich K. Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J Mol Biol. 1991 Feb 5;217(3):517–530. doi: 10.1016/0022-2836(91)90754-t. [DOI] [PubMed] [Google Scholar]
  10. Havel T. F. An evaluation of computational strategies for use in the determination of protein structure from distance constraints obtained by nuclear magnetic resonance. Prog Biophys Mol Biol. 1991;56(1):43–78. doi: 10.1016/0079-6107(91)90007-f. [DOI] [PubMed] [Google Scholar]
  11. Hayashi T., Zushi M., Yamamoto S., Suzuki K. Further localization of binding sites for thrombin and protein C in human thrombomodulin. J Biol Chem. 1990 Nov 25;265(33):20156–20159. [PubMed] [Google Scholar]
  12. Huang L. H., Cheng H., Pardi A., Tam J. P., Sweeney W. V. Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX. Biochemistry. 1991 Jul 30;30(30):7402–7409. doi: 10.1021/bi00244a006. [DOI] [PubMed] [Google Scholar]
  13. Højrup P., Magnusson S. Disulphide bridges of bovine factor X. Biochem J. 1987 Aug 1;245(3):887–891. doi: 10.1042/bj2450887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  15. Lentz S. R., Chen Y., Sadler J. E. Sequences required for thrombomodulin cofactor activity within the fourth epidermal growth factor-like domain of human thrombomodulin. J Biol Chem. 1993 Jul 15;268(20):15312–15317. [PubMed] [Google Scholar]
  16. Lougheed J. C., Bowman C. L., Meininger D. P., Komives E. A. Thrombin inhibition by cyclic peptides from thrombomodulin. Protein Sci. 1995 Apr;4(4):773–780. doi: 10.1002/pro.5560040417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  18. Montelione G. T., Wüthrich K., Nice E. C., Burgess A. W., Scheraga H. A. Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain-fold by nuclear magnetic resonance and distance geometry. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5226–5230. doi: 10.1073/pnas.84.15.5226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moy F. J., Li Y. C., Rauenbuehler P., Winkler M. E., Scheraga H. A., Montelione G. T. Solution structure of human type-alpha transforming growth factor determined by heteronuclear NMR spectroscopy and refined by energy minimization with restraints. Biochemistry. 1993 Jul 27;32(29):7334–7353. doi: 10.1021/bi00080a003. [DOI] [PubMed] [Google Scholar]
  20. Nagashima M., Lundh E., Leonard J. C., Morser J., Parkinson J. F. Alanine-scanning mutagenesis of the epidermal growth factor-like domains of human thrombomodulin identifies critical residues for its cofactor activity. J Biol Chem. 1993 Feb 5;268(4):2888–2892. [PubMed] [Google Scholar]
  21. Ni F., Ripoll D. R., Purisima E. O. Conformational stability of a thrombin-binding peptide derived from the hirudin C-terminus. Biochemistry. 1992 Mar 10;31(9):2545–2554. doi: 10.1021/bi00124a015. [DOI] [PubMed] [Google Scholar]
  22. Nilges M., Clore G. M., Gronenborn A. M. Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms. Circumventing problems associated with folding. FEBS Lett. 1988 Oct 24;239(1):129–136. doi: 10.1016/0014-5793(88)80559-3. [DOI] [PubMed] [Google Scholar]
  23. Padmanabhan K., Padmanabhan K. P., Tulinsky A., Park C. H., Bode W., Huber R., Blankenship D. T., Cardin A. D., Kisiel W. Structure of human des(1-45) factor Xa at 2.2 A resolution. J Mol Biol. 1993 Aug 5;232(3):947–966. doi: 10.1006/jmbi.1993.1441. [DOI] [PubMed] [Google Scholar]
  24. Savage C. R., Jr, Hash J. H., Cohen S. Epidermal growth factor. Location of disulfide bonds. J Biol Chem. 1973 Nov 25;248(22):7669–7672. [PubMed] [Google Scholar]
  25. Selander-Sunnerhagen M., Ullner M., Persson E., Teleman O., Stenflo J., Drakenberg T. How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X. J Biol Chem. 1992 Sep 25;267(27):19642–19649. doi: 10.2210/pdb1ccf/pdb. [DOI] [PubMed] [Google Scholar]
  26. Smith B. O., Downing A. K., Dudgeon T. J., Cunningham M., Driscoll P. C., Campbell I. D. Secondary structure of fibronectin type 1 and epidermal growth factor modules from tissue-type plasminogen activator by nuclear magnetic resonance. Biochemistry. 1994 Mar 8;33(9):2422–2429. doi: 10.1021/bi00175a010. [DOI] [PubMed] [Google Scholar]
  27. Srinivasan J., Hu S., Hrabal R., Zhu Y., Komives E. A., Ni F. Thrombin-bound structure of an EGF subdomain from human thrombomodulin determined by transferred nuclear Overhauser effects. Biochemistry. 1994 Nov 22;33(46):13553–13560. doi: 10.1021/bi00250a007. [DOI] [PubMed] [Google Scholar]
  28. Stearns D. J., Kurosawa S., Esmon C. T. Microthrombomodulin. Residues 310-486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation. J Biol Chem. 1989 Feb 25;264(6):3352–3356. [PubMed] [Google Scholar]
  29. Wen D. Z., Dittman W. A., Ye R. D., Deaven L. L., Majerus P. W., Sadler J. E. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene. Biochemistry. 1987 Jul 14;26(14):4350–4357. doi: 10.1021/bi00388a025. [DOI] [PubMed] [Google Scholar]
  30. Wishart D. S., Sykes B. D., Richards F. M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry. 1992 Feb 18;31(6):1647–1651. doi: 10.1021/bi00121a010. [DOI] [PubMed] [Google Scholar]
  31. Ye J., Esmon N. L., Esmon C. T., Johnson A. E. The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation. J Biol Chem. 1991 Dec 5;266(34):23016–23021. [PubMed] [Google Scholar]

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