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. 1999 Oct;77(4):2237–2250. doi: 10.1016/S0006-3495(99)77064-5

Properties of spin and fluorescent labels at a receptor-ligand interface.

R Owenius 1, M Osterlund 1, M Lindgren 1, M Svensson 1, O H Olsen 1, E Persson 1, P O Freskgård 1, U Carlsson 1
PMCID: PMC1300504  PMID: 10512843

Abstract

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.

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Selected References

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  1. Banner D. W., D'Arcy A., Chène C., Winkler F. K., Guha A., Konigsberg W. H., Nemerson Y., Kirchhofer D. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature. 1996 Mar 7;380(6569):41–46. doi: 10.1038/380041a0. [DOI] [PubMed] [Google Scholar]
  2. Davie E. W., Fujikawa K., Kisiel W. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry. 1991 Oct 29;30(43):10363–10370. doi: 10.1021/bi00107a001. [DOI] [PubMed] [Google Scholar]
  3. Freskgård P. O., Olsen O. H., Persson E. Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy. Protein Sci. 1996 Aug;5(8):1531–1540. doi: 10.1002/pro.5560050809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gibbs C. S., McCurdy S. N., Leung L. L., Paborsky L. R. Identification of the factor VIIa binding site on tissue factor by homologous loop swap and alanine scanning mutagenesis. Biochemistry. 1994 Nov 29;33(47):14003–14010. doi: 10.1021/bi00251a007. [DOI] [PubMed] [Google Scholar]
  5. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  6. Hammarström P., Kalman B., Jonsson B. H., Carlsson U. Pyrene excimer fluorescence as a proximity probe for investigation of residual structure in the unfolded state of human carbonic anhydrase II. FEBS Lett. 1997 Dec 22;420(1):63–68. doi: 10.1016/s0014-5793(97)01488-9. [DOI] [PubMed] [Google Scholar]
  7. Harlos K., Martin D. M., O'Brien D. P., Jones E. Y., Stuart D. I., Polikarpov I., Miller A., Tuddenham E. G., Boys C. W. Crystal structure of the extracellular region of human tissue factor. Nature. 1994 Aug 25;370(6491):662–666. doi: 10.1038/370662a0. [DOI] [PubMed] [Google Scholar]
  8. Heiden W., Moeckel G., Brickmann J. A new approach to analysis and display of local lipophilicity/hydrophilicity mapped on molecular surfaces. J Comput Aided Mol Des. 1993 Oct;7(5):503–514. doi: 10.1007/BF00124359. [DOI] [PubMed] [Google Scholar]
  9. Hubbell W. L., Gross A., Langen R., Lietzow M. A. Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol. 1998 Oct;8(5):649–656. doi: 10.1016/s0959-440x(98)80158-9. [DOI] [PubMed] [Google Scholar]
  10. Kelley R. F., Costas K. E., O'Connell M. P., Lazarus R. A. Analysis of the factor VIIa binding site on human tissue factor: effects of tissue factor mutations on the kinetics and thermodynamics of binding. Biochemistry. 1995 Aug 22;34(33):10383–10392. doi: 10.1021/bi00033a009. [DOI] [PubMed] [Google Scholar]
  11. Klug C. S., Eaton S. S., Eaton G. R., Feix J. B. Ligand-induced conformational change in the ferric enterobactin receptor FepA as studied by site-directed spin labeling and time-domain ESR. Biochemistry. 1998 Jun 23;37(25):9016–9023. doi: 10.1021/bi980144e. [DOI] [PubMed] [Google Scholar]
  12. Lindgren M., Svensson M., Freskgård P. O., Carlsson U., Jonasson P., Mårtensson L. G., Jonsson B. H. Characterization of a folding intermediate of human carbonic anhydrase II: probing local mobility by electron paramagnetic resonance. Biophys J. 1995 Jul;69(1):202–213. doi: 10.1016/S0006-3495(95)79892-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mann K. G., Nesheim M. E., Church W. R., Haley P., Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood. 1990 Jul 1;76(1):1–16. [PubMed] [Google Scholar]
  14. Mchaourab H. S., Kálai T., Hideg K., Hubbell W. L. Motion of spin-labeled side chains in T4 lysozyme: effect of side chain structure. Biochemistry. 1999 Mar 9;38(10):2947–2955. doi: 10.1021/bi9826310. [DOI] [PubMed] [Google Scholar]
  15. Muller Y. A., Ultsch M. H., Kelley R. F., de Vos A. M. Structure of the extracellular domain of human tissue factor: location of the factor VIIa binding site. Biochemistry. 1994 Sep 13;33(36):10864–10870. doi: 10.1021/bi00202a003. [DOI] [PubMed] [Google Scholar]
  16. Muller Y. A., Ultsch M. H., de Vos A. M. The crystal structure of the extracellular domain of human tissue factor refined to 1.7 A resolution. J Mol Biol. 1996 Feb 16;256(1):144–159. doi: 10.1006/jmbi.1996.0073. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Persson E., Olsen O. H., Ostergaard A., Nielsen L. S. Ca2+ binding to the first epidermal growth factor-like domain of factor VIIa increases amidolytic activity and tissue factor affinity. J Biol Chem. 1997 Aug 8;272(32):19919–19924. doi: 10.1074/jbc.272.32.19919. [DOI] [PubMed] [Google Scholar]
  19. Persson M., Hammarström P., Lindgren M., Jonsson B. H., Svensson M., Carlsson U. EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: evidence for increased flexibility of the hydrophobic core by the interaction. Biochemistry. 1999 Jan 5;38(1):432–441. doi: 10.1021/bi981442e. [DOI] [PubMed] [Google Scholar]
  20. Richardson J. S. The anatomy and taxonomy of protein structure. Adv Protein Chem. 1981;34:167–339. doi: 10.1016/s0065-3233(08)60520-3. [DOI] [PubMed] [Google Scholar]
  21. Riddles P. W., Blakeley R. L., Zerner B. Reassessment of Ellman's reagent. Methods Enzymol. 1983;91:49–60. doi: 10.1016/s0076-6879(83)91010-8. [DOI] [PubMed] [Google Scholar]
  22. Ruf W., Kelly C. R., Schullek J. R., Martin D. M., Polikarpov I., Boys C. W., Tuddenham E. G., Edgington T. S. Energetic contributions and topographical organization of ligand binding residues of tissue factor. Biochemistry. 1995 May 16;34(19):6310–6315. doi: 10.1021/bi00019a008. [DOI] [PubMed] [Google Scholar]
  23. Ruf W., Schullek J. R., Stone M. J., Edgington T. S. Mutational mapping of functional residues in tissue factor: identification of factor VII recognition determinants in both structural modules of the predicted cytokine receptor homology domain. Biochemistry. 1994 Feb 15;33(6):1565–1572. doi: 10.1021/bi00172a037. [DOI] [PubMed] [Google Scholar]
  24. Schullek J. R., Ruf W., Edgington T. S. Key ligand interface residues in tissue factor contribute independently to factor VIIa binding. J Biol Chem. 1994 Jul 29;269(30):19399–19403. [PubMed] [Google Scholar]
  25. Stone M. J., Ruf W., Miles D. J., Edgington T. S., Wright P. E. Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization. Biochem J. 1995 Sep 1;310(Pt 2):605–614. doi: 10.1042/bj3100605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Svensson M., Jonasson P., Freskgård P. O., Jonsson B. H., Lindgren M., Mårtensson L. G., Gentile M., Borén K., Carlsson U. Mapping the folding intermediate of human carbonic anhydrase II. Probing substructure by chemical reactivity and spin and fluorescence labeling of engineered cysteine residues. Biochemistry. 1995 Jul 11;34(27):8606–8620. doi: 10.1021/bi00027a010. [DOI] [PubMed] [Google Scholar]
  27. Thim L., Bjoern S., Christensen M., Nicolaisen E. M., Lund-Hansen T., Pedersen A. H., Hedner U. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry. 1988 Oct 4;27(20):7785–7793. doi: 10.1021/bi00420a030. [DOI] [PubMed] [Google Scholar]
  28. Zhang E., St Charles R., Tulinsky A. Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant. J Mol Biol. 1999 Feb 5;285(5):2089–2104. doi: 10.1006/jmbi.1998.2452. [DOI] [PubMed] [Google Scholar]

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