TM-1是從台灣龜殼花分離出來的一種小分子蛇毒蛋白酶，它晶體結構的解析度到1.84Å，經改變後所獲得的R和Rfree 個別是0.179和0.216。TM-1的整體結構是呈現一個扁橢圓形狀，在整個TM-1結構中擁有三個雙硫鍵，分別是在Cys119-Cys198、Cys160-Cys182、Cys162-Cys165的位置上。整個TM-1結構中，裡面有一個zinc ion被四個配體(ligand)所包圍，這四個配體(ligand)包含了三個hisditines和一個水分子，它們形成了tetrahedral geometry的構形。我們發現TM-1和TM-3之間活性位置結構的差異可能顯示了TM-1和TM-3對內生性的抑制劑有不同的敏感性。再者，我們已經藉由塑造的方式把三個內生性的抑制劑(pEKW、pEQW、pENW)放入到TM-1的活性結構位置。我們從抑制劑和TM-1相互作用力的分析結果可以看出抑制劑P-2位置的氨基酸可以和TM-1形成氫鍵和恐水性的作用力，所以抑制劑P-2位置對TM-1的鍵結是很重要的。藉由氧化胰島素B鏈對TM-的蛋白質水解的實驗，我們猜測基質的P-1和P-2位置對TM-1的切割是很重要的。P-1位置所偏好的氨基酸是Leu、Phe和His，而P-2位置所偏好的氨基酸是Leu、Val和Tyr。在這些結論中,我們的結果指示TM-1和TM-3結構的差異可能影響它們對抑制劑的敏感性和基質專一性。除此之外，我們也猜測基質的P-1和P-2位置對TM-1切割基質是重要的。

The crystal structure of TM-1, a small snake-venom metalloproteinase (SVMP) isolated from Taiwan habu (Trimeresurus mucrosquamatus), was determined at 1.84 Å resolution with resultant R and Rfree values of 0.179 and 0.216, respectively. The overall structure of TM-1 is an oblate ellipsoid that contains three disulfide crosslinks, Cys119-Cys198, Cys160-Cys182 and Cys162-Cys165. The overall structure of TM-1 contains one zinc ion which is bound to four ligands, including three conserved histidines and one water molecule, displaying a tetrahedral geometry. We find that the distinct active site structures between TM-1 and TM-3 may reflect the different sensitivities toward the endogenous inhibitors from structural comparison of TM-1 and TM-3. Moreover, we have modeled the three endogenous inhibitors, i.e., pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) and pyroGlu-Lys-Trp (pEKW), into the active site of TM-1 structure. Results from interaction analysis of TM-1 and inhibitors show that the P-2 site of inhibitors is important for binding to TM-1 via hydrogen bonds and hydrophobic interactions. By proteolysis experiments of TM-1 using oxidized insulin B-chain as substrate, we sugguest P-1 and P-2 sites of substrates are important for cleavage by TM-1. The preferential amino acids at P-1 site are Leu, Phe and His, while The preferential amino acids at P-2 site are Leu, Val and Tyr. In conclusions, our results indicate that the structural difference between TM-1 and TM-3 may influence their endogenous inhibitor sensitivities and substrate specificity. Additionally, we also suggest that the P-1 and P-2 sites of substrate are critical for cleavage by TM-1.

Baramova, E.N., Shannon, J.D., Bjarnason, J.B., and Fox, J.W. (1989). Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Archives of Biochemistry and Biophysics 275, 63-71.
Baramova, E.N., Shannon, J.D., Bjarnason, J.B., and Fox, J.W. (1990). Identification of the cleavage sites by a hemorrhagic metalloproteinase in type IV collagen. Matrix (Stuttgart, Germany) 10, 91-97.
Bjarnason, J.B., Hamilton, D., and Fox, J.W. (1988). Studies on the mechanism of hemorrhage production by five proteolytic hemorrhagic toxins from Crotalus atrox venom. Biological Chemistry Hoppe-Seyler 369 Suppl, 121-129.
Bode, W., Grams, F., Reinemer, P., Gomis-Ruth, F.X., Baumann, U., McKay, D.B., and Stocker, W. (1996). The metzincin-superfamily of zinc-peptidases. Advances in Experimental Medicine and Biology 389, 1-11.
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905-921.
Catanese, J.J., and Kress, L.F. (1992). Isolation from opossum serum of a metalloproteinase inhibitor homologous to human alpha 1B-glycoprotein. Biochemistry 31, 410-418.
Engh, R. A. & Huber, R. (1991). Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallogr A Biol Crystallogr 47, 392-400.
Francis, B., and Kaiser, II (1993). Inhibition of metalloproteinases in Bothrops asper venom by endogenous peptides. Toxicon 31, 889-899.
Francis, B., Seebart, C., and Kaiser, II (1992). Citrate is an endogenous inhibitor of snake venom enzymes by metal-ion chelation. Toxicon 30, 1239-1246.
Freitas, M.A., Geno, P.W., Sumner, L.W., Cooke, M.E., Hudiburg, S.A., Ownby, C.L., Kaiser, II, and Odell, G.V. (1992). Citrate is a major component of snake venoms. Toxicon 30, 461-464.
Gomis-Ruth, F.X., Kress, L.F., and Bode, W. (1993). First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/collagenases. The European Molecular Biology Organization Journal 12, 4151-4157.
Gomis-Ruth, F.X., Kress, L.F., Kellermann, J., Mayr, I., Lee, X., Huber, R., and Bode, W. (1994). Refined 2.0 A X-ray crystal structure of the snake venom zinc-endopeptidase adamalysin II. Primary and tertiary structure determination, refinement, molecular structure and comparison with astacin, collagenase and thermolysin. Journal of Molecular Biology 239, 513-544.
Gong, W., Zhu, X., Liu, S., Teng, M., and Niu, L. (1998). Crystal structures of acutolysin A, a three-disulfide hemorrhagic zinc metalloproteinase from the snake venom of Agkistrodon acutus. Journal of Molecular Biology 283, 657-668.
Grams, F., Huber, R., Kress, L.F., Moroder, L., and Bode, W. (1993). Activation of snake venom metalloproteinases by a cysteine switch-like mechanism. Federation of European Biochemical Societies Letters 335, 76-80.
Gutierrez, J.M., and Rucavado, A. (2000). Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage. Biochimie 82, 841-850.
Hite, L.A., Shannon, J.D., Bjarnason, J.B., and Fox, J.W. (1992). Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures. Biochemistry 31, 6203-6211.
Huang, K.F., Chiou, S.H., Ko, T.P., Yuann, J.M., and Wang, A.H. (2002). The 1.35 A structure of cadmium-substituted TM-3, a snake-venom metalloproteinase from Taiwan habu: elucidation of a TNFalpha-converting enzyme-like active-site structure with a distorted octahedral geometry of cadmium. Acta Crystallogr D Biol Crystallogr 58, 1118-1128.
Huang, K.F., Chow, L.P., and Chiou, S.H. (1999). Isolation and characterization of a novel proteinase inhibitor from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus). Biochemical and Biophysical Research Communications 263, 610-616.
Huang, K.F., Hung, C.C., and Chiou, S.H. (1993). Characterization of three fibrinogenolytic proteases isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus). Biochemistry and Molecular Biology International 31, 1041-1050.
Huang, K.F., Hung, C.C., Pan, F.M., Chow, L.P., Tsugita, A., and Chiou, S.H. (1995). Characterization of multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus mucrosquamatus): protein microsequencing coupled with cDNA sequence analysis. Biochemical and Biophysical Research Communications 216, 223-233.
Huang, K.F., Hung, C.C., Wu, S.H., and Chiou, S.H. (1998). Characterization of three endogenous peptide inhibitors for multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus mucrosquamatus). Biochemical and Biophysical Research Communications 248, 562-568.
Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallographica 47 ( Pt 2), 110-119.
Kato, H., Iwanaga, S., and Suzuki, T. (1966). The isolation and amino acid sequences of new pyroglutamylpeptides from snake venoms. Experientia 22, 49-50.
Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M.(1993). PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography 26, 283-291.
Mebs, D. (1998) Enzymes in snake venom: an overview.In Enzymes from Snake Venom (Bailey, G.S., ed.), pp. 1–10. Alaken,Inc., Colorado.
Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Crystallogr A Biol Crystallogr 50, 157-163.
Neves-Ferreira, A.G., Perales, J., Fox, J.W., Shannon, J.D., Makino, D.L., Garratt, R.C., and Domont, G.B. (2002). Structural and functional analyses of DM43, a snake venom metalloproteinase inhibitor from Didelphis marsupialis serum. The Journal of Biological Chemistry 277, 13129-13137.
Odell, G.V., Ferry, P.C., Vick, L.M., Fenton, A.W., Decker, L.S., Cowell, R.L., Ownby, C.L., and Gutierrez, J.M. (1998). Citrate inhibition of snake venom roteases. Toxicon 36, 1801-1806.
Otwinowski, Z. & Minor,W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology 276, 307-326.
Randolph, A., Chamberlain, S.H., Chu, H.L., Retzios, A.D., Markland, F.S., Jr., and Masiarz, F.R. (1992). Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. Protein Science 1, 590-600.
Robeva, A., Politi, V., Shannon, J.D., Bjarnason, J.B., and Fox, J.W. (1991). Synthetic and endogenous inhibitors of snake venom metalloproteinases. Biomedica Biochimica Acta 50, 769-773.
Rodrigues, V.M., Soares, A.M., Guerra-Sa, R., Rodrigues, V., Fontes, M.R., and Giglio, J.R. (2000). Structural and functional characterization of neuwiedase, a nonhemorrhagic fibrin(ogen)olytic metalloprotease from Bothrops neuwiedi snake venom. Archives of Biochemistry and Biophysics 381, 213-224.
Shannon, J.D., Baramova, E.N., Bjarnason, J.B., and Fox, J.W. (1989). Amino acid sequence of a Crotalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin. The Journal of Biological Chemistry 264, 11575-11583.
Stocker, W., and Bode, W. (1995). Structural features of a superfamily of zinc-endopeptidases: the metzincins. Current Opinion in Structural Biology 5, 383-390.
Stocker, W., Grams, F., Baumann, U., Reinemer, P., Gomis-Ruth, F.X., McKay, D.B., and Bode, W. (1995). The metzincins--topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein Science 4, 823-840.
Takeya, H., Onikura, A., Nikai, T., Sugihara, H., and Iwanaga, S. (1990). Primary structure of a hemorrhagic metalloproteinase, HT-2, isolated from the venom of Crotalus ruber ruber. Journal of Biochemistry 108, 711-719.
Takeya, H. & Iwanaga, S. (1998) Proteases that induce hemorrhage. In Enzymes from Snake Venom (Bailey, G.S., ed.), pp.11–38.Alaken, Inc., Colorado.
Tu, A.T., Baker, B., Wongvibulsin, S., and Willis, T. (1996). Biochemical characterization of atroxase and nucleotide sequence encoding the fibrinolytic enzyme. Toxicon 34, 1295-1300.
Valente, R.H., Dragulev, B., Perales, J., Fox, J.W., and Domont, G.B. (2001). BJ46a, a snake venom metalloproteinase inhibitor. Isolation, characterization, cloning and insights into its mechanism of action. European Journal of Biochemistry / Federation of European Biochemical Societies 268, 3042-3052.
Watanabe, L., Shannon, J.D., Valente, R.H., Rucavado, A., Alape-Giron, A., Kamiguti, A.S., Theakston, R.D., Fox, J.W., Gutierrez, J.M., and Arni, R.K. (2003). Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities. Protein Science 12, 2273-2281.
Yamakawa, Y., and Omori-Satoh, T. (1992). Primary structure of the antihemorrhagic factor in serum of the Japanese Habu: a snake venom metalloproteinase inhibitor with a double-headed cystatin domain. Journal of Biochemistry 112, 583-589.
Zhang, D., Botos, I., Gomis-Ruth, F.X., Doll, R., Blood, C., Njoroge, F.G., Fox, J.W., Bode, W., and Meyer, E.F. (1994). Structural interaction of natural and synthetic inhibitors with the venom metalloproteinase, atrolysin C (form d). Proceedings of the National Academy of Sciences of the United States of America 91, 8447-8451.