Cooperative binding of calcium ions modulates the tertiary structure and catalytic activity of Matrix-Metalloproteinase-9


To ascertain the molecular basis of Ca2+-mediated activation of matrix metalloproteinase-9 (MMP-9), we determined the accessibility of tryptophan residues to externally added acrylamide as quencher in the absence and presence of the metal ion. The steady-state and time resolved fluorescence data revealed that MMP-9 possesses two classes of tryptophan residues, “exposed” and “buried” which are quenched by the collisional rate constants (kq) of 3.2′ 109M-1.s-1 and 7.5′ 108M-1.s-1, respectively. These values are impaired by approximately two and three-fold, respectively, in the presence of 10 mM Ca2+. The Stern-Volmer constants (Ksv values) predicted from the time resolved fluorescence data (in the absence of Ca2+ ) satisfied the dynamic quenching model of the enzyme’s tryptophan residues. This was not the case in the presence of Ca2+ ; the steady-state acrylamide quenching data could only be explained by a combination of “dynamic” and “static” quenching models. A cumulative account of these data led to the suggestion that the binding of Ca2+ modulated the tertiary structure of the protein by decreasing the dynamic flexibility of the enzyme, which is manifested in further structuring of the enzyme’s active site pocket toward facilitating catalysis. Arguments are presented that the binding of Ca2+ at distal sites “dynamically” communicates with the
active site residues of MMP-9 during catalysis.

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Tobwala, S. and Srivastava, D. (2013) Cooperative binding of calcium ions modulates the tertiary structure and catalytic activity of Matrix-Metalloproteinase-9. Advances in Enzyme Research, 1, 17-29. doi: 10.4236/aer.2013.12002.

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The authors declare no conflicts of interest.


[1] Chou, K.C., Kahari, V.M. and Saarialho-Kere, U. (1997) Matrix metalloproteinases in skin. Experimental Dermatology, 6, 199-213. doi:10.1111/j.1600-0625.1997.tb00164.x PMid:9450622
[2] Saarialho-Kere, U.K. (1998) Patterns of matrix metalloproteinase and TIMP expression in chronic ulcers. Archives of Dermatological Research, 290, S47-S54. doi:10.1007/PL00007453 PMid:9710383
[3] Nagase, H. and Woessner Jr., J.F. (1999) Matrix metalloproteinases. Journal of Biological Chemistry, 274, 21491-21494. doi:10.1074/jbc.274.31.21491 PMid:10419448
[4] Kerkela, E. and Saarialho-Kere, U. (2003) Matrix metalloproteinases in tumor progression: Focus on basal and squamous cell skin cancer. Experimental Dermatology, 12, 109-125. doi:10.1034/j.1600-0625.2003.120201.x PMid:12702139
[5] Nelson, A.R., Fingleton, B., Rothenberg, M.L. and Matrisian, L.M. (2000) Matrix metalloproteinases: Biologic activity and clinical implications. Journal of Clinical Oncology, 18, 1135-1149.
[6] Liotta, L.A., Tryggvason, K., Garbisa, S., Hart, I., Foltz, C.M. and Shafie, S. (1980) Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature, 284, 67-68. doi:10.1038/284067a0 PMid:6243750
[7] Polette, M., Gilbert, N., Stas, I., Nawrocki, B., Noel, A., Remacle, A., Stetler-Stevenson, W.G., Birembaut, P. and Foidart, M. (1994) Gelatinase A expression and localization in human breast cancers. An in situ hybridization study and immunohistochemical detection using confocal microscopy. Virchows Archives, 424, 641-645. doi:10.1007/BF00195779
[8] Dalberg, K., Eriksson, E., Enberg, U., Kjellman, M. and Backdahl, M. (2004) Gelatinase A, membrane type 1 matrix metalloproteinase, and extracellular matrix metalloproteinase inducer mRNA expression: correlation with invasive growth of breast cancer. World Journal of Surgery, 24, 334-340. doi:10.1007/s002689910053 PMid:10658069
[9] Hanemaaijer, R., Verheijen, J.H., Maguire, T.M., Visser, H., Toet, K., McDermott, E., O’Higgins, N. and Duffy, M.J. (2000) Increased gelatinase-A and gelatinase-B activities in malignant vs. benign breast tumors. International Journal of Cancer, 86, 204-207. doi:10.1002/(SICI)1097-0215(20000415)86:2<204::AID-IJC9>3.0.CO;2-6
[10] Papadopoulou, S., Scorilas, A., Arnogianaki, N., Papapanayiotou, B., Tzimogiani, A., Agnantis, N. and Talieri, M. (2001) Expression of gelatinase-A (MMP-2) in human colon cancer and normal colon mucosa. Tumor Biology, 22, 383-389. doi:10.1159/000050641 PMid:11786732
[11] Segain, J.P., Harb, J., Gregoire, M., Meflah, K. and Menanteau, J. (1996) Induction of fibroblast gelatinase B expression by direct contact with cell lines derived from primary tumor but not from metastases. Cancer Research, 56, 5506-5512.
[12] Tokuraku, M., Sato, H., Murakami, S., Okada, Y., Watanabe, Y. and Seiki, M. (1995) Activation of the precursor of gelatinase A/72 kDa type IV collagenase/MMP-2 in lung carcinomas correlates with the expression of membrane-type matrix metalloproteinase (MT-MMP) and with lymph node metastasis. International Journal of Cancer, 64, 355-359. doi:10.1002/ijc.2910640513 PMid:7591310
[13] Nagawa, H. and Yagihashi, S. (1994) Expression of type IV collagen and its degrading enzymes in squamous cell carcinoma of lung. Japanese Journal of Cancer Research, 85, 934-938. doi:10.1111/j.1349-7006.1994.tb02971.x
[14] Sehgal, G., Hua, J., Bernhard, E.J., Sehgal, I., Thompson, T.C. and Muschel, R.J. (1998) Requirement for matrix metalloproteinase-9 (gelatinase B) expression in metastasis by murine prostate carcinoma. American Journal of Pathology, 152, 591-596.
[15] Koshiba, T., Hosotani, R., Wada, M., Fujimoto, K., Lee, J.U., Doi, R., Arii, S. and Imamura, M. (1997) Detection of matrix metalloproteinase activity in human pancreatic cancer. Surgery Today, 27, 302-304. doi:10.1007/BF00941802 PMid:9086544
[16] Gress, T.M., Muller-Pillasch, F., Lerch, M.M., Friess, H., Buchler, M. and Adler, G. (1995) Expression and insitu localization of genes coding for extracellular matrix proteins and extracellular matrix degrading proteases in pancreatic cancer. International Journal of Cancer, 62, 407-413. doi:10.1002/ijc.2910620409 PMid:7635566
[17] Young, T.N., Rodriguez, G.C., Rinehart, A.R., Bast Jr., R.C., Pizzo, S.V. and Stack, M.S. (1996) Characterization of gelatinases linked to extracellular matrix invasion in ovarian adenocarcinoma: Purification of matrix metallo-proteinase 2. Gynecologic Oncology, 62, 89-99. doi:10.1006/gyno.1996.0195 PMid:8690299
[18] Hsieh, M.S., Ho, H.C., Chou, D.T., Pan, S., Liang, Y.C., Hsieh, T.Y., Lan, J.L. and Tsai, S.H. (2003) Expression of matrix metalloproteinase-9 (gelatinase B) in gouty arthritis and stimulation of MMP-9 by urate crystals in macrophages. Journal of Cellular Biochemistry, 89, 791-799. doi:10.1002/jcb.10530 PMid:12858344
[19] Pirila, E., Ramamurthy, N.S., Sorsa, T., Salo, T., Hietanen, J. and Maisi, P. (2003) Gelatinase A (MMP-2), collagenase-2 (MMP-8), and laminin-5 gamma2-chain expression in murine inflammatory bowel disease (ulcerative colitis). Digestive Diseases Sciences, 48, 93-98. doi:10.1023/A:1021790532723
[20] Pyo, R., Lee, J.K., Shipley, J.M., Curci, J.A., Mao, D., Ziporin, S.J., Ennis, T.L., Shapiro, S.D., Senior, R.M. and Thompson, R.W. (2000) Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms. Journal of Clinical Investigation, 105, 1641-1649. doi:10.1172/JCI8931
[21] Kossakowska, A.E., Medlicott, S.A., Edwards, D.R., Guyn, L., Stabbler, A.L., Sutherland, L.R. and Urbanski, S.J. (1999) Elevated plasma gelatinase A (MMP-2) activity is associated with quiescent Crohn’s Disease. Annals of the New York Academy of Sciences, 878, 578-580. doi:10.1111/j.1749-6632.1999.tb07732.x PMid:10415778
[22] Kee, C., Son, S. and Ahn, B. (1999) The relationship between gelatinase A activity in aqueous humor and glaucoma. Journal of Glaucoma, 8, 51-55. doi:10.1097/00061198-199902000-00011
[23] Fisher, G.J. and Kang, S. (2002) Phototherapy for scleroderma: Biologic rationale, results, and promise. Current Opinion in Rheumatology, 14, 723-726. doi:10.1097/00002281-200211000-00016
[24] Overall, C.M. and Lopez-Otin, C. (2002) Strategies for MMP inhibition in cancer: Innovations for the post-trial era. Nature Reviews Cancer, 2, 657-672. doi:10.1038/nrc884
[25] Bode, W., Fernandez-Catalan, C., Tschesche, H., Grams, F., Nagase, H. and Maskos, K. (1999) Structural properties of matrix metalloproteinases. Cellular and Molecular Life Sciences, 55, 639-652. doi:10.1007/s000180050320
[26] Rowsell, S., Hawtin, P., Minshull, C.A., Jepson, H., Brockbank, S.M., Barratt, D.G., Slater, A.M., McPheat, W.L., Waterson, D., Henney, A.M. and Pauptit, R.A. (2002) Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor. Journal of Molecular Biology, 319, 173-181. doi:10.1016/S0022-2836(02)00262-0
[27] Elkins, P.A., Ho, Y.S., Smith, W.W., Janson, C.A., D’Alessio, K.J., McQueney, M.S., Cummings, M.D. and Romanic, A.M. (2002) Structure of the C-terminally truncated human ProMMP9, a gelatin-binding matrix metalloproteinase. Acta Crystallographica Section D, 58, 1182- 1192. doi:10.1107/S0907444902007849
[28] Tochowicz, A., Maskos, K., Huber, R., Oltenfreiter, R., Dive, V., Yiotakis, A., Zanda, M., Pourmotabbed, T., Bode, W. and Goettig, P. (2007) Crystal structures of MMP-9 complexes with five inhibitors: Contribution of the flexible Arg424 side-chain to selectivity. Journal of Molecular Biology, 371, 989-1006. doi:10.1016/j.jmb.2007.05.068
[29] Zhang, Y., Dean, W.L. and Gray, R.D. (1997) Cooperative binding of Ca2+ to human interstitial collagenase assessed by circular dichroism, fluorescence, and catalytic activity. Journal of Molecular Biology, 272, 1444-1447.
[30] Lowry, C.L., McGeehan, G. and LeVine, H. (1992) Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase. Proteins, 12, 42-48. doi:10.1002/prot.340120106
[31] Housley, T.J., Baumann, A.P., Braun, I.D., Davis, G., Seperack, P.K. and Wilhelm, S.M. (1993) Recombinant Chinese hamster ovary cell matrix metalloprotease-3 (MMP-3, stromelysin-1). Role of calcium in promatrix metalloprotease-3 (pro-MMP-3, prostromelysin-1) activation and thermostability of the low mass catalytic domain of MMP-3. Journal of Biological Chemistry, 268, 4481-4487.
[32] Lee, S., Park, H.I. and Sang, Q.X. (2007) Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion. Biochemical Journal, 403, 31-42. doi:10.1042/BJ20061390
[33] Gossas, T. and Danielson, U.H. (2006) Characterization of Ca2+ interactions with matrix metallopeptidase-12: Implications for matrix metallopeptidase regulation. Biochemical Journal, 398, 393-398. doi:10.1042/BJ20051933
[34] Wetmore, D.R. and Hardman, K.D. (1996) Roles of the propeptide and metal ions in the folding and stability of the catalytic domain of stromelysin (matrix metalloproteinase 3). Biochemistry, 35, 6549-6558. doi:10.1021/bi9530752
[35] Diaz, N. and Suarez, D. (2007) Molecular dynamics simulations of matrix metalloproteinase 2: Role of the structural metal ions. Biochemistry, 46, 8943-8952. doi:10.1021/bi700541p
[36] Munshi, H.G., Wu, Y.I., Ariztia, E.V. and Stack, M.S. (2002) Calcium regulation of matrix metalloproteinase-mediated migration in oral squamous cell carcinoma cells. Journal of Biological Chemistry, 277, 41480-41488. doi:10.1074/jbc.M207695200
[37] Winberg, J.O., Berg, E., Kolset, S.O. and Uhlin-Hansen, L. (2003) Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans. European Journal of Biochemistry, 270, 3996-4007. doi:10.1046/j.1432-1033.2003.03788.x
[38] Cheng, D., Shen, Q., Nan, F., Qian, Z. and Ye, Q.Z. (2003) Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for highthroughput inhibitor screening. Protein Expression and Purification, 27, 63-74. doi:10.1016/S1046-5928(02)00530-2
[39] Hammes-Schiffer, S. (2006) Hydrogen tunneling and protein motion in enzyme reactions. Accounts of Chemical Research, 39, 93-100. doi:10.1021/ar040199a
[40] Eftink, M.R. and Ghiron, C.A. (1976) Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry, 15, 672-680. doi:10.1021/bi00648a035
[41] Eftink, M.R. and Ghiron, C.A. (1981) Fluorescence quenching studies with proteins. Analytical Biochemistry, 114, 199-227. doi:10.1016/0003-2697(81)90474-7
[42] Chang, Y.C. and Ludescher, R.D. (1993) Tryptophan fluorescence quenching in rabbit skeletal myosin rod. Biophysical Chemistry, 48, 49-59. doi:10.1016/0301-4622(93)80041-G
[43] Chao, P.W. and Chow, C.S. (2007) Monitoring aminoglycoside-induced conformational changes in 16S rRNA through acrylamide quenching. Bioorganic & Medicinal Chemistry, 15, 3825-3831. doi:10.1016/j.bmc.2007.03.025
[44] Khan, K.K., Ma-zumdar, S., Modi, S., Sutcliffe, M., Roberts, G.C. and Mitra, S. (1997) Steady-state and picosecond-time-resolved fluorescence studies on the recombinant heme domain of Bacillus megaterium cytochrome P-450. European Journal of Biochemistry, 244, 361-370. doi:10.1111/j.1432-1033.1997.00361.x
[45] Stack, M.S., Itoh, Y., Young, T.N. and Nagase, H. (1996) Fluorescence quenching studies of matrix metalloproteinases (MMPs): Evidence for structural rearrangement of the pro-MMP-2/TIMP-2 complex upon mercurial activation. Archives of Biochemistry and Biophysics, 333, 163-169. doi:10.1006/abbi.1996.0377
[46] Sambrook, J., Fritsch, E.F. and Maniatis, T. (2000) Molecular cloning: A la-boratory manual. Cold Spring Harbor Press, New York.
[47] Szabo, J. and Rayner, D. (1980) Fluorescence decay of tryptophan conformers in aqueous solution. Journal of American Chemical Society, 102, 554-563. doi:10.1021/ja00522a020
[48] Stobiecka, A. (2005) Acrylamide-quenching of Rhizomucor miehei lipase. Journal of Photochemistry and Photobiology B: Biology, 80, 9-18. doi:10.1016/j.jphotobiol.2005.02.002
[49] Becker, A., Schlichting, I., Kabsch, W., Groche, D., Schultz, S. and Wagner, A.F. (1998) Iron center, substrate recognition and mechanism of peptide deformylase. Nature Structural & Molecular Biology, 5, 1053-1058. doi:10.1038/4162
[50] Dardel, F., Ragusa, S., Lazennec, C., Blanquet, S. and Meinnel, T. (1998) Solution structure of nickel-peptide deformylase. Journal of Molecular Biology, 280, 501-513. doi:10.1006/jmbi.1998.1882

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