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EMBO J. Jun 1990; 9(6): 1939–1947.
PMCID: PMC551902

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.

Abstract

A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta-sheet wrapped around a five turn alpha-helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal 'trunk' (occupying the 'unprimed' subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.

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Selected References

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  • Abe K, Emori Y, Kondo H, Arai S, Suzuki K. The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli. J Biol Chem. 1988 Jun 5;263(16):7655–7659. [PubMed]
  • Abrahamson M, Ritonja A, Brown MA, Grubb A, Machleidt W, Barrett AJ. Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J Biol Chem. 1987 Jul 15;262(20):9688–9694. [PubMed]
  • Anastasi A, Brown MA, Kembhavi AA, Nicklin MJ, Sayers CA, Sunter DC, Barrett AJ. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J. 1983 Apr 1;211(1):129–138. [PMC free article] [PubMed]
  • Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, Hanada K. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189–198. [PMC free article] [PubMed]
  • Bernstein FC, Koetzle TF, Williams GJ, Meyer EF, Jr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. [PubMed]
  • Björk I, Ylinenjärvi K. Interaction of chicken cystatin with inactivated papains. Biochem J. 1989 May 15;260(1):61–68. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Chaiken IM, Smith EL. Reaction of the sulfhydryl group of papain with chloroacetic acid. J Biol Chem. 1969 Oct 10;244(19):5095–5099. [PubMed]
  • Drenth J, Jansonius JN, Koekoek R, Wolthers BG. The structure of papain. Adv Protein Chem. 1971;25:79–115. [PubMed]
  • Green GD, Kembhavi AA, Davies ME, Barrett AJ. Cystatin-like cysteine proteinase inhibitors from human liver. Biochem J. 1984 Mar 15;218(3):939–946. [PMC free article] [PubMed]
  • Järvinen M, Rinne A. Human spleen cysteineproteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants. Biochim Biophys Acta. 1982 Nov 9;708(2):210–217. [PubMed]
  • Jerala R, Trstenjak M, Lenarcic B, Turk V. Cloning a synthetic gene for human stefin B and its expression in E. coli. FEBS Lett. 1988 Oct 24;239(1):41–44. [PubMed]
  • Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. [PubMed]
  • Kamphuis IG, Kalk KH, Swarte MB, Drenth J. Structure of papain refined at 1.65 A resolution. J Mol Biol. 1984 Oct 25;179(2):233–256. [PubMed]
  • Lenney JF, Tolan JR, Sugai WJ, Lee AG. Thermostable endogenous inhibitors of cathepsins B and H. Eur J Biochem. 1979 Nov 1;101(1):153–161. [PubMed]
  • 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. [PubMed]
  • Nikawa T, Towatari T, Ike Y, Katunuma N. Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants. Evidence that the QVVAG region is not essential for cysteine proteinase inhibitory activities. FEBS Lett. 1989 Sep 25;255(2):309–314. [PubMed]
  • Ohkubo I, Kurachi K, Takasawa T, Shiokawa H, Sasaki M. Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry. 1984 Nov 20;23(24):5691–5697. [PubMed]
  • Ritonja A, Machleidt W, Barrett AJ. Amino acid sequence of the intracellular cysteine proteinase inhibitor cystatin B from human liver. Biochem Biophys Res Commun. 1985 Sep 30;131(3):1187–1192. [PubMed]
  • Sluyterman LA, Wijdenes J. An agarose mercurial column for the separation of mercaptopapain and nonmercaptopapain. Biochim Biophys Acta. 1970 Mar 31;200(3):593–595. [PubMed]
  • Teno N, Tsuboi S, Itoh N, Okamoto H, Okada Y. Significant effects of Z-Gln-Val-Val-OME, common sequences of thiol proteinase inhibitors on thiol proteinases. Biochem Biophys Res Commun. 1987 Mar 13;143(2):749–752. [PubMed]
  • Thiele U, Auerswald EA, Gebhard W, Assfalg-Machleidt I, Popović T, Machleidt W. Inhibitorily active recombinant human stefin B. Gene synthesis, expression and isolation of an inhibitory active MS-2 pol-stefin B fusion protein and preparation of Des[Met1,2(2)]stefin B. Biol Chem Hoppe Seyler. 1988 Oct;369(10):1167–1178. [PubMed]

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