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. 1995 Dec;4(12):2487–2498. doi: 10.1002/pro.5560041205

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

S R Van Doren 1, A V Kurochkin 1, W Hu 1, Q Z Ye 1, L L Johnson 1, D J Hupe 1, E R Zuiderweg 1
PMCID: PMC2143039  PMID: 8580839

Abstract

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.

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Selected References

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  1. Birkedal-Hansen H., Moore W. G., Bodden M. K., Windsor L. J., Birkedal-Hansen B., DeCarlo A., Engler J. A. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4(2):197–250. doi: 10.1177/10454411930040020401. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Gomis-Rüth F. X., Huber R., Zwilling R., Stöcker W. Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases. Nature. 1992 Jul 9;358(6382):164–167. doi: 10.1038/358164a0. [DOI] [PubMed] [Google Scholar]
  3. Bode W., Reinemer P., Huber R., Kleine T., Schnierer S., Tschesche H. The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity. EMBO J. 1994 Mar 15;13(6):1263–1269. doi: 10.1002/j.1460-2075.1994.tb06378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Borkakoti N., Winkler F. K., Williams D. H., D'Arcy A., Broadhurst M. J., Brown P. A., Johnson W. H., Murray E. J. Structure of the catalytic domain of human fibroblast collagenase complexed with an inhibitor. Nat Struct Biol. 1994 Feb;1(2):106–110. doi: 10.1038/nsb0294-106. [DOI] [PubMed] [Google Scholar]
  5. Clavel C., Polette M., Doco M., Binninger I., Birembaut P. Immunolocalization of matrix metallo-proteinases and their tissue inhibitor in human mammary pathology. Bull Cancer. 1992;79(3):261–270. [PubMed] [Google Scholar]
  6. 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]
  7. Darlak K., Miller R. B., Stack M. S., Spatola A. F., Gray R. D. Thiol-based inhibitors of mammalian collagenase. Substituted amide and peptide derivatives of the leucine analogue, 2-[(R,S)-mercaptomethyl]-4-methylpentanoic acid. J Biol Chem. 1990 Mar 25;265(9):5199–5205. [PubMed] [Google Scholar]
  8. DiPasquale G., Caccese R., Pasternak R., Conaty J., Hubbs S., Perry K. Proteoglycan- and collagen-degrading enzymes from human interleukin 1-stimulated chondrocytes from several species: proteoglycanase and collagenase inhibitors as potentially new disease-modifying antiarthritic agents. Proc Soc Exp Biol Med. 1986 Nov;183(2):262–267. doi: 10.3181/00379727-183-42416. [DOI] [PubMed] [Google Scholar]
  9. Firestein G. S., Paine M. M. Stromelysin and tissue inhibitor of metalloproteinases gene expression in rheumatoid arthritis synovium. Am J Pathol. 1992 Jun;140(6):1309–1314. [PMC free article] [PubMed] [Google Scholar]
  10. Gooley P. R., Johnson B. A., Marcy A. I., Cuca G. C., Salowe S. P., Hagmann W. K., Esser C. K., Springer J. P. Secondary structure and zinc ligation of human recombinant short-form stromelysin by multidimensional heteronuclear NMR. Biochemistry. 1993 Dec 7;32(48):13098–13108. doi: 10.1021/bi00211a020. [DOI] [PubMed] [Google Scholar]
  11. Grobelny D., Poncz L., Galardy R. E. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry. 1992 Aug 11;31(31):7152–7154. doi: 10.1021/bi00146a017. [DOI] [PubMed] [Google Scholar]
  12. Gronenborn A. M., Clore G. M. Identification of N-terminal helix capping boxes by means of 13C chemical shifts. J Biomol NMR. 1994 May;4(3):455–458. doi: 10.1007/BF00179351. [DOI] [PubMed] [Google Scholar]
  13. Grzesiek S., Bax A. Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomol NMR. 1993 Mar;3(2):185–204. doi: 10.1007/BF00178261. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hu P., Ye Q. Z., Loo J. A. Calcium stoichiometry determination for calcium binding proteins by electrospray ionization mass spectrometry. Anal Chem. 1994 Dec 1;66(23):4190–4194. doi: 10.1021/ac00095a013. [DOI] [PubMed] [Google Scholar]
  16. Hyberts S. G., Goldberg M. S., Havel T. F., Wagner G. The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X-ray structures. Protein Sci. 1992 Jun;1(6):736–751. doi: 10.1002/pro.5560010606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li J., Brick P., O'Hare M. C., Skarzynski T., Lloyd L. F., Curry V. A., Clark I. M., Bigg H. F., Hazleman B. L., Cawston T. E. Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller. Structure. 1995 Jun 15;3(6):541–549. doi: 10.1016/s0969-2126(01)00188-5. [DOI] [PubMed] [Google Scholar]
  18. Lovejoy B., Cleasby A., Hassell A. M., Longley K., Luther M. A., Weigl D., McGeehan G., McElroy A. B., Drewry D., Lambert M. H. Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor. Science. 1994 Jan 21;263(5145):375–377. doi: 10.1126/science.8278810. [DOI] [PubMed] [Google Scholar]
  19. Lovejoy B., Hassell A. M., Luther M. A., Weigl D., Jordan S. R. Crystal structures of recombinant 19-kDa human fibroblast collagenase complexed to itself. Biochemistry. 1994 Jul 12;33(27):8207–8217. doi: 10.1021/bi00193a006. [DOI] [PubMed] [Google Scholar]
  20. Matrisian L. M. The matrix-degrading metalloproteinases. Bioessays. 1992 Jul;14(7):455–463. doi: 10.1002/bies.950140705. [DOI] [PubMed] [Google Scholar]
  21. Nanzer A. P., Poulsen F. M., van Gunsteren W. F., Torda A. E. A reassessment of the structure of chymotrypsin inhibitor 2 (CI-2) using time-averaged NMR restraints. Biochemistry. 1994 Dec 6;33(48):14503–14511. doi: 10.1021/bi00252a017. [DOI] [PubMed] [Google Scholar]
  22. Neri D., Szyperski T., Otting G., Senn H., Wüthrich K. Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry. 1989 Sep 19;28(19):7510–7516. doi: 10.1021/bi00445a003. [DOI] [PubMed] [Google Scholar]
  23. Netzel-Arnett S., Fields G. B., Birkedal-Hansen H., Van Wart H. E., Fields G. Sequence specificities of human fibroblast and neutrophil collagenases. J Biol Chem. 1991 Apr 15;266(11):6747–6755. [PubMed] [Google Scholar]
  24. Niedzwiecki L., Teahan J., Harrison R. K., Stein R. L. Substrate specificity of the human matrix metalloproteinase stromelysin and the development of continuous fluorometric assays. Biochemistry. 1992 Dec 22;31(50):12618–12623. doi: 10.1021/bi00165a011. [DOI] [PubMed] [Google Scholar]
  25. Okada Y., Shinmei M., Tanaka O., Naka K., Kimura A., Nakanishi I., Bayliss M. T., Iwata K., Nagase H. Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium. Lab Invest. 1992 Jun;66(6):680–690. [PubMed] [Google Scholar]
  26. Okada Y., Takeuchi N., Tomita K., Nakanishi I., Nagase H. Immunolocalization of matrix metalloproteinase 3 (stromelysin) in rheumatoid synovioblasts (B cells): correlation with rheumatoid arthritis. Ann Rheum Dis. 1989 Aug;48(8):645–653. doi: 10.1136/ard.48.8.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reinemer P., Grams F., Huber R., Kleine T., Schnierer S., Piper M., Tschesche H., Bode W. Structural implications for the role of the N terminus in the 'superactivation' of collagenases. A crystallographic study. FEBS Lett. 1994 Jan 31;338(2):227–233. doi: 10.1016/0014-5793(94)80370-6. [DOI] [PubMed] [Google Scholar]
  28. Salowe S. P., Marcy A. I., Cuca G. C., Smith C. K., Kopka I. E., Hagmann W. K., Hermes J. D. Characterization of zinc-binding sites in human stromelysin-1: stoichiometry of the catalytic domain and identification of a cysteine ligand in the proenzyme. Biochemistry. 1992 May 19;31(19):4535–4540. doi: 10.1021/bi00134a001. [DOI] [PubMed] [Google Scholar]
  29. Sasaguri Y., Komiya S., Sugama K., Suzuki K., Inoue A., Morimatsu M., Nagase H. Production of matrix metalloproteinases 2 and 3 (stromelysin) by stromal cells of giant cell tumor of bone. Am J Pathol. 1992 Sep;141(3):611–621. [PMC free article] [PubMed] [Google Scholar]
  30. Shima I., Sasaguri Y., Kusukawa J., Yamana H., Fujita H., Kakegawa T., Morimatsu M. Production of matrix metalloproteinase-2 and metalloproteinase-3 related to malignant behavior of esophageal carcinoma. A clinicopathologic study. Cancer. 1992 Dec 15;70(12):2747–2753. doi: 10.1002/1097-0142(19921215)70:12<2747::aid-cncr2820701204>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  31. Stams T., Spurlino J. C., Smith D. L., Wahl R. C., Ho T. F., Qoronfleh M. W., Banks T. M., Rubin B. Structure of human neutrophil collagenase reveals large S1' specificity pocket. Nat Struct Biol. 1994 Feb;1(2):119–123. doi: 10.1038/nsb0294-119. [DOI] [PubMed] [Google Scholar]
  32. Stetler-Stevenson W. G., Aznavoorian S., Liotta L. A. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol. 1993;9:541–573. doi: 10.1146/annurev.cb.09.110193.002545. [DOI] [PubMed] [Google Scholar]
  33. Van Doren S. R., Kurochkin A. V., Ye Q. Z., Johnson L. L., Hupe D. J., Zuiderweg E. R. Assignments for the main-chain nuclear magnetic resonances and delineation of the secondary structure of the catalytic domain of human stromelysin-1 as obtained from triple-resonance 3D NMR experiments. Biochemistry. 1993 Dec 7;32(48):13109–13122. doi: 10.1021/bi00211a021. [DOI] [PubMed] [Google Scholar]
  34. Walakovits L. A., Moore V. L., Bhardwaj N., Gallick G. S., Lark M. W. Detection of stromelysin and collagenase in synovial fluid from patients with rheumatoid arthritis and posttraumatic knee injury. Arthritis Rheum. 1992 Jan;35(1):35–42. doi: 10.1002/art.1780350106. [DOI] [PubMed] [Google Scholar]
  35. Whitham S. E., Murphy G., Angel P., Rahmsdorf H. J., Smith B. J., Lyons A., Harris T. J., Reynolds J. J., Herrlich P., Docherty A. J. Comparison of human stromelysin and collagenase by cloning and sequence analysis. Biochem J. 1986 Dec 15;240(3):913–916. doi: 10.1042/bj2400913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilhelm S. M., Shao Z. H., Housley T. J., Seperack P. K., Baumann A. P., Gunja-Smith Z., Woessner J. F., Jr Matrix metalloproteinase-3 (stromelysin-1). Identification as the cartilage acid metalloprotease and effect of pH on catalytic properties and calcium affinity. J Biol Chem. 1993 Oct 15;268(29):21906–21913. [PubMed] [Google Scholar]
  37. Woessner J. F., Jr Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991 May;5(8):2145–2154. [PubMed] [Google Scholar]
  38. Yamazaki T., Yoshida M., Nagayama K. Complete assignments of magnetic resonances of ribonuclease H from Escherichia coli by double- and triple-resonance 2D and 3D NMR spectroscopies. Biochemistry. 1993 Jun 1;32(21):5656–5669. doi: 10.1021/bi00072a023. [DOI] [PubMed] [Google Scholar]
  39. Ye Q. Z., Johnson L. L., Hupe D. J., Baragi V. Purification and characterization of the human stromelysin catalytic domain expressed in Escherichia coli. Biochemistry. 1992 Nov 17;31(45):11231–11235. doi: 10.1021/bi00160a038. [DOI] [PubMed] [Google Scholar]
  40. Ye Q. Z., Johnson L. L., Nordan I., Hupe D., Hupe L. A recombinant human stromelysin catalytic domain identifying tryptophan derivatives as human stromelysin inhibitors. J Med Chem. 1994 Jan 7;37(1):206–209. doi: 10.1021/jm00027a027. [DOI] [PubMed] [Google Scholar]

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