Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 Nov;9(11):2218–2224. doi: 10.1110/ps.9.11.2218

The N-terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two-step binding by rapid-kinetic studies of cystatin A labeled at the N-terminus with a fluorescent reporter group.

S Estrada 1, S T Olson 1, E Raub-Segall 1, I Björk 1
PMCID: PMC2144488  PMID: 11152132

Abstract

The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two-step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N-terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS- and AEDANS-labeled cystatin A to papain resulted in approximately 4-fold and 1.2-fold increases of probe fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS-cystatin A increased linearly with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin-proteinase reactions.

Full Text

The Full Text of this article is available as a PDF (152.5 KB).

Selected References

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

  1. Abrahamson M., Mason R. W., Hansson H., Buttle D. J., Grubb A., Ohlsson K. Human cystatin C. role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase. Biochem J. 1991 Feb 1;273(Pt 3):621–626. doi: 10.1042/bj2730621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auerswald E. A., Nägler D. K., Schulze A. J., Engh R. A., Genenger G., Machleidt W., Fritz H. Production, inhibitory activity, folding and conformational analysis of an N-terminal and an internal deletion variant of chicken cystatin. Eur J Biochem. 1994 Sep 1;224(2):407–415. doi: 10.1111/j.1432-1033.1994.00407.x. [DOI] [PubMed] [Google Scholar]
  3. Björk I., Alriksson E., Ylinenjärvi K. Kinetics of binding of chicken cystatin to papain. Biochemistry. 1989 Feb 21;28(4):1568–1573. doi: 10.1021/bi00430a022. [DOI] [PubMed] [Google Scholar]
  4. Björk I., Brieditis I., Abrahamson M. Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases. Biochem J. 1995 Mar 1;306(Pt 2):513–518. doi: 10.1042/bj3060513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Björk I., Brieditis I., Raub-Segall E., Pol E., Håkansson K., Abrahamson M. The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases. Biochemistry. 1996 Aug 20;35(33):10720–10726. doi: 10.1021/bi960420u. [DOI] [PubMed] [Google Scholar]
  6. Björk I., Pol E., Raub-Segall E., Abrahamson M., Rowan A. D., Mort J. S. Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes. Biochem J. 1994 Apr 1;299(Pt 1):219–225. doi: 10.1042/bj2990219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Björk I., Ylinenjärvi K. Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin. Biochemistry. 1990 Feb 20;29(7):1770–1776. doi: 10.1021/bi00459a016. [DOI] [PubMed] [Google Scholar]
  8. Bode W., Engh R., Musil D., Thiele U., Huber R., Karshikov A., Brzin J., Kos J., Turk V. The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J. 1988 Aug;7(8):2593–2599. doi: 10.1002/j.1460-2075.1988.tb03109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dieckmann T., Mitschang L., Hofmann M., Kos J., Turk V., Auerswald E. A., Jaenicke R., Oschkinat H. The structures of native phosphorylated chicken cystatin and of a recombinant unphosphorylated variant in solution. J Mol Biol. 1993 Dec 20;234(4):1048–1059. doi: 10.1006/jmbi.1993.1658. [DOI] [PubMed] [Google Scholar]
  10. Ekiel I., Abrahamson M., Fulton D. B., Lindahl P., Storer A. C., Levadoux W., Lafrance M., Labelle S., Pomerleau Y., Groleau D. NMR structural studies of human cystatin C dimers and monomers. J Mol Biol. 1997 Aug 15;271(2):266–277. doi: 10.1006/jmbi.1997.1150. [DOI] [PubMed] [Google Scholar]
  11. Estrada S., Nycander M., Hill N. J., Craven C. J., Waltho J. P., Björk I. The role of Gly-4 of human cystatin A (stefin A) in the binding of target proteinases. Characterization by kinetic and equilibrium methods of the interactions of cystatin A Gly-4 mutants with papain, cathepsin B, and cathepsin L. Biochemistry. 1998 May 19;37(20):7551–7560. doi: 10.1021/bi980026r. [DOI] [PubMed] [Google Scholar]
  12. Estrada S., Pavlova A., Björk I. The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L. Biochemistry. 1999 Jun 1;38(22):7339–7345. doi: 10.1021/bi990003s. [DOI] [PubMed] [Google Scholar]
  13. Fujishima A., Imai Y., Nomura T., Fujisawa Y., Yamamoto Y., Sugawara T. The crystal structure of human cathepsin L complexed with E-64. FEBS Lett. 1997 Apr 21;407(1):47–50. doi: 10.1016/s0014-5793(97)00216-0. [DOI] [PubMed] [Google Scholar]
  14. Guncar G., Pungercic G., Klemencic I., Turk V., Turk D. Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S. EMBO J. 1999 Feb 15;18(4):793–803. doi: 10.1093/emboj/18.4.793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hall A., Abrahamson M., Grubb A., Trojnar J., Kania P., Kasprzykowska R., Kasprzykowski F. Cystatin C based peptidyl diazomethanes as cysteine proteinase inhibitors: influence of the peptidyl chain length. J Enzyme Inhib. 1992;6(2):113–123. doi: 10.3109/14756369209040742. [DOI] [PubMed] [Google Scholar]
  16. Hall A., Håkansson K., Mason R. W., Grubb A., Abrahamson M. Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases. J Biol Chem. 1995 Mar 10;270(10):5115–5121. doi: 10.1074/jbc.270.10.5115. [DOI] [PubMed] [Google Scholar]
  17. Kuzmic P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem. 1996 Jun 1;237(2):260–273. doi: 10.1006/abio.1996.0238. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lindahl P., Abrahamson M., Björk I. Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin. Biochem J. 1992 Jan 1;281(Pt 1):49–55. doi: 10.1042/bj2810049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lindahl P., Alriksson E., Jörnvall H., Björk I. Interaction of the cysteine proteinase inhibitor chicken cystatin with papain. Biochemistry. 1988 Jul 12;27(14):5074–5082. doi: 10.1021/bi00414a019. [DOI] [PubMed] [Google Scholar]
  21. Lindahl P., Nycander M., Ylinenjärvi K., Pol E., Björk I. Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin. Biochem J. 1992 Aug 15;286(Pt 1):165–171. doi: 10.1042/bj2860165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Machleidt W., Thiele U., Laber B., Assfalg-Machleidt I., Esterl A., Wiegand G., Kos J., Turk V., Bode W. Mechanism of inhibition of papain by chicken egg white cystatin. Inhibition constants of N-terminally truncated forms and cyanogen bromide fragments of the inhibitor. FEBS Lett. 1989 Jan 30;243(2):234–238. doi: 10.1016/0014-5793(89)80135-8. [DOI] [PubMed] [Google Scholar]
  23. Martin J. R., Craven C. J., Jerala R., Kroon-Zitko L., Zerovnik E., Turk V., Waltho J. P. The three-dimensional solution structure of human stefin A. J Mol Biol. 1995 Feb 17;246(2):331–343. doi: 10.1006/jmbi.1994.0088. [DOI] [PubMed] [Google Scholar]
  24. McGrath M. E., Klaus J. L., Barnes M. G., Brömme D. Crystal structure of human cathepsin K complexed with a potent inhibitor. Nat Struct Biol. 1997 Feb;4(2):105–109. doi: 10.1038/nsb0297-105. [DOI] [PubMed] [Google Scholar]
  25. McGrath M. E., Palmer J. T., Brömme D., Somoza J. R. Crystal structure of human cathepsin S. Protein Sci. 1998 Jun;7(6):1294–1302. doi: 10.1002/pro.5560070604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Musil D., Zucic D., Turk D., Engh R. A., Mayr I., Huber R., Popovic T., Turk V., Towatari T., Katunuma N. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J. 1991 Sep;10(9):2321–2330. doi: 10.1002/j.1460-2075.1991.tb07771.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nycander M., Estrada S., Mort J. S., Abrahamson M., Björk I. Two-step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop. FEBS Lett. 1998 Jan 23;422(1):61–64. doi: 10.1016/s0014-5793(97)01604-9. [DOI] [PubMed] [Google Scholar]
  28. Pol E., Olsson S. L., Estrada S., Prasthofer T. W., Björk I. Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases. Biochem J. 1995 Oct 1;311(Pt 1):275–282. doi: 10.1042/bj3110275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schreiber G., Fersht A. R. Rapid, electrostatically assisted association of proteins. Nat Struct Biol. 1996 May;3(5):427–431. doi: 10.1038/nsb0596-427. [DOI] [PubMed] [Google Scholar]
  30. Stubbs M. T., Laber B., Bode W., Huber R., Jerala R., Lenarcic B., Turk V. The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J. 1990 Jun;9(6):1939–1947. doi: 10.1002/j.1460-2075.1990.tb08321.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Turk B., Krizaj I., Turk V. Isolation and characterization of bovine stefin B. Biol Chem Hoppe Seyler. 1992 Jul;373(7):441–446. doi: 10.1515/bchm3.1992.373.2.441. [DOI] [PubMed] [Google Scholar]
  32. Turk B., Stoka V., Björk I., Boudier C., Johansson G., Dolenc I., Colic A., Bieth J. G., Turk V. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen. Protein Sci. 1995 Sep;4(9):1874–1880. doi: 10.1002/pro.5560040922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Turk B., Turk V., Turk D. Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. Biol Chem. 1997 Mar-Apr;378(3-4):141–150. [PubMed] [Google Scholar]
  34. Turk V., Bode W. The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett. 1991 Jul 22;285(2):213–219. doi: 10.1016/0014-5793(91)80804-c. [DOI] [PubMed] [Google Scholar]
  35. Zhao B., Janson C. A., Amegadzie B. Y., D'Alessio K., Griffin C., Hanning C. R., Jones C., Kurdyla J., McQueney M., Qiu X. Crystal structure of human osteoclast cathepsin K complex with E-64. Nat Struct Biol. 1997 Feb;4(2):109–111. doi: 10.1038/nsb0297-109. [DOI] [PubMed] [Google Scholar]

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

RESOURCES