Journal of Protein Chemistry

, Volume 16, Issue 6, pp 637–650 | Cite as

Expression, Purification, Characterization, and X-Ray Analysis of Selenomethionine 215 Variant of Leukocyte Collagenase

  • Michael Pieper
  • Michael Betz
  • Nediljko Budisa
  • Franz-Xaver Gomis-Rüth
  • Wolfram Bode
  • Harald Tschesche


Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et al. (1993) FEBS331, 134–140; Stöcker et al. (1995) Protein Sci.4, 823–840]. The distal S–CH3 moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80–Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by Selenomethionine. Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry. The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in changed kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon urea denaturation. The X-ray crystal structure of this Selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.

Matrix metalloproteinases Met-turn Selenomethionine conformational stability X-ray crystallography 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Baumann, U., Wu, S., Flaherty, K. M., and McKay, D. B. (1993). EMBO J. 12, 3357–3364.Google Scholar
  2. Beiboer, S. H. W., Van den Berg, D., Dekker, N., Cox, R. C., and Verheij, H. M. (1996). Protein Eng. 9, 345–352.Google Scholar
  3. Bernhard, A. R., Wells, R. N. C., Cleasby, A., Borlat, F., Payton, M. A., and Proudfoot, A. E. I. (1995). Eur. J. Biochem. 230, 111–118.Google Scholar
  4. Bode, W., Gomis-Rüth, F.-X., and Stöcker, W. (1993). FEBS 331, 134–140.Google Scholar
  5. Bode, W., Reinemer, P., Huber, R., Kleine, T., Schnierer, S., and Tschesche, H. (1994). EMBO 13, 1263–1269.Google Scholar
  6. Boles, J. O., Cisneros, R. J., Weir, M. S., Odom, J. D., Villafranca, J. E., and Dunlap, R. B. (1991). Biochemistry 30, 11073–11080.Google Scholar
  7. Boles, J. O., Tolleson, W. H., Schmidt, J. C., Dunlap, R. B., and Odom, J. D. (1992). J. Biol. Chem. 267, 22217–22223.Google Scholar
  8. Brünger, A. T. (1991). Curr. Opin. Struct. Biol. 1, 1016–1022.Google Scholar
  9. Budisa, N., Steipe, B., Demange, P., Eckerskorn, C., Kellermann, J., and Huber, R. (1995). Eur. J. Biochem. 230, 788–796.Google Scholar
  10. CCP4. (1994). Acta Crystallogr. D 50, 760–763.Google Scholar
  11. Cornish-Bowden, A., and Eisenthal, R. (1978). Biochem. Biophys. Acta 523, 268–272.Google Scholar
  12. Covey, T. R., Bonner, R. F., Shushan, B. I., and Henion, J. D. (1988). Rapid Commun. Mass. Spectrom. 2, 249–256.Google Scholar
  13. Cowie, D. B., and Cohen, G. N. (1957). Biochim. Biophys. Acta 26, 252–261.Google Scholar
  14. Engh, R. A., and Huber, R. (1991). Acta Crystallogr. A 47, 392–400.Google Scholar
  15. Frank, P., Licht, A., Tullis, T. B. D., Hodgson, K. O., and Pecht, I. (1985). J. Biol. Chem. 260, 5518–5525.Google Scholar
  16. Gellman, S. H. (1991). Biochemistry 30, 6633–6636.Google Scholar
  17. Gill, S. C., and von Hippel, P. H. (1989). Analyt. Biochem. 182, 319–326.Google Scholar
  18. Hädener, A., Matzinger, P. K., Malashkevich, V. M., Louie, G. V., Wood, S. P., Oliver, P., Alefounder, P. R., Pitt, A. R., Abell, C., and Battersby, A. R. (1993). Eur. J. Biochem. 211, 615–624.Google Scholar
  19. Hendrickson, W. A., Horton, J. R., and LeMaster, D. M. (1990). EMBO 9, 1665–1672.Google Scholar
  20. Huber, R. E., and Criddle, R. S. (1967). Biochim. Biophys. Acta 141, 587–599.Google Scholar
  21. Kleine, T., Bartsch, S., Bläser, J., Schnierer, S., Triebel, S., Valentin, M., Gote, T., and Tschesche, H. (1993). Biochemistry 32, 14125–14131.Google Scholar
  22. Knight, C. G., Willenbrock, F., and Murphy, G. (1992). FEBS 296, 263–266.Google Scholar
  23. Kraulis, P. J. (1991). J. Appl. Crystallogr. 24, 946–950.Google Scholar
  24. Laemmli, U. K. (1970). Nature 227, 680–685.Google Scholar
  25. Leslie, A. G. W. (1991). In Crystallographic Computering V (Moras, D., Podjarny, A. D., and Thierry, J. C.,, eds), Oxford University Press, Oxford, pp. 27–38.Google Scholar
  26. Mann, M., and Wilm, M. (1995). TIBS 20, 219–224.Google Scholar
  27. Mann, M., Meng, C. K., and Fenn, J. A. (1989). Anal. Chem. 61, 1702–1708.Google Scholar
  28. Matrisian, L. M. (1990). TIG 6, 121–125.Google Scholar
  29. Moore, W. M., and Spilburg, C. A. (1986). Biochemistry 25, 5189–5195.Google Scholar
  30. Murphy, G., and Docherty, A. J. P. (1992). Am. J. Cell. Biol. 7, 120–125.Google Scholar
  31. O'Neil, K. T., and DeGrado, W. F. (1990). TIBS 15, 59–64.Google Scholar
  32. Pace, C. N. (1986). Meth. Enzymol. 131, 266–280.Google Scholar
  33. Pace, C. N., Shirley, B. A., and Thomson, J. A. (1989). In Protein Structure: A Practical Approach (Creighton, T. E., ed.), IRL, pp. 311–330.Google Scholar
  34. Puente, X. S., Pendas, A. M., Leano, E., Velasco, G., and Lopez-Otin, C. (1996). Cancer Res. 56, 944–949.Google Scholar
  35. Qoronfleh, M. W., Ho, T. F., Brake, P. G., Banks, T. M., Pulvino, T. A., Wahl, R. C., Eshraghi, J., Chowdhury, S. K., Ciccarelli, R. B., and Jones, B. N. (1995). J. Biotech. 39, 119–128.Google Scholar
  36. Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Pieper, M., Tschesche, H., and Bode, W. (1994). FEBS 338, 227–233.Google Scholar
  37. Richards, F. M., and Lim, W. A. (1994). Q. Rev. Biophys. 26, 423–498.Google Scholar
  38. Ries, C., and Petrides, P. E. (1995). Biol. Chem. Hoppe-Seyler 376, 345–355.Google Scholar
  39. Rose, G. D., Geselowitz, A. R., Lesser, G. J., Lee, R. H., and Zehfus, M. H. (1985). Science 229, 834–838.Google Scholar
  40. Sandberg, W. S., and Terwilliger, T. C. (1989). Science 245, 54–57.Google Scholar
  41. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). Proc. Natl. Acad. Sci. USA 74, 5463–5467.Google Scholar
  42. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994). Nature 370, 61–65.Google Scholar
  43. Schnierer, S., Kleine, T., Gote, T., Hillemann, A., Knäuper, V., and Tschesche, H. (1993). Biochem. Biophys. Res. Commun. 191, 319–326.Google Scholar
  44. Stöcker, W., Grams, F., Baumann, U., Reinemer, P., Gomis-Rüth, F.-X., McKay, D. B., and Bode, W. (1995). Protein Sci. 4, 823–840.Google Scholar
  45. Studier, F. W., and Moffat, B. (1986). J. Mol. Biol. 189, 113–130.Google Scholar
  46. Studier, W. F., Rosenberg, A. H., Dunn, J. J. and Durbendorff, A. (1990). Methods Enzymol. 185, 60–89.Google Scholar
  47. Studier, F. W., et al. 1990.Google Scholar
  48. Takino, T., Sato, H., Shinagawa, A., and Seiki, M. (1995). J. Biol. Chem. 270, 23013–23020.Google Scholar
  49. Wallace, C. J. A., and Clark-Lewis, I. (1992). J. Biol. Chem. 267, 3852–3861.Google Scholar
  50. Will, B., and Hinzmann, B. (1995). Eur. J. Biochem. 231, 602–608.Google Scholar
  51. Woessner, J. F., Jr. (1991). FASEB J. 5, 2145–2154.Google Scholar
  52. Yang, W., Hendrickson, W. A., Crouch, R. J., and Satow, Y. (1990). Science 249, 1398–1405.Google Scholar

Copyright information

© Plenum Publishing Corporation 1997

Authors and Affiliations

  • Michael Pieper
    • 1
  • Michael Betz
    • 2
  • Nediljko Budisa
    • 2
  • Franz-Xaver Gomis-Rüth
    • 2
  • Wolfram Bode
    • 2
  • Harald Tschesche
    • 1
  1. 1.Fakultät für Chemie und BiochemieUniversität BielefeldBielefeldGermany
  2. 2.Abteilung fur StrukturforschungMax-Planck-Institut für BiochemieMartinsriedGermany

Personalised recommendations