心臟毒素A6為台灣眼鏡蛇蛇毒的成份，我們發現到台灣眼鏡蛇在東岸黑腹與西岸白腹的台灣眼鏡蛇蛇毒成份有些許的不同，而心臟毒素A6主要是在東岸黑腹的眼鏡蛇中含量最高；在這篇研究中，從結構中發現了幾個結果：(一)我們可以從心臟毒素A6的晶體結構中，可以發現到A6與Tγ (Toxin γ Nigricoliis)的晶體結構在指環區I的Pro8與Pro9呈現相同的順式(cis)結構，而與Tγ的NMR結構中在指環區I的Pro8與Pro9所呈現的反式(trans)結構不同；這或許提供了我們對於心臟毒素在水溶液中與晶體結構中指環區I在Pro8與Pro9所形成結構不同的興趣。(二)而在A6的指環區II中也發現到鍵結有兩個結合水，並且形成氫鍵，發現與已知結構的心臟毒素也同樣鍵結有結合水，只是在鍵結的結合水數目上有所不同，與不同的氨基酸形成氫鍵。(三)而我們也發現到A6與Tγ在晶體堆疊上有類似之處。這些在結構中發現的結果，或許會影響心臟毒素結構在與細胞膜產生反應時的模式。

Cardiotoxin VI (CTX A6) is one component of Taiwan cobra venom. Cardiotoxin is different from component of Taiwan cobra venom between eastern and western Taiwan cobra. And CTX A6 is from eastern Taiwan cobra. In this study, we have found (1) From the crystal structure of CTX A6, Pro8 and Pro9 at loop I in CTX A6 and Tγ appear to have the same cis- conformation, but the structures of Pro8 and Pro9 at loop I in CTX A6 is different from the NMR structure of CTX Tγ. The difference of the structures of Pro8 and Pro9 at loop I in CTX A6 and Tγ may affect the interaction of Cardiotoxins and membrane. (2) In the crystal structure of CTX A6, there are two bound waters in loop II forming three hydrogen bonds. In other Cardiotoxins(CTX A3、Tγ、A5、O2), there are also bound waters in loop II. (3) In crystal packing of CTX A6 and Tγ, there are some similarities in terms of trimer orientation. Therefore, these findings from the crystal structure of CTX A6 at high-resolution 1.6Å may imply the interaction between Cardiotoxins and membrane.

1. Herz , J. M., Johnson , D. A. and Taylor, P. (1989) Distance between the agonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor. J.Biol.Chem., 264 , 12439-12448.
2. Changeux, J. P., Kasai, M. and Lee, C. Y. (1970) Use of snake venom toxins to characterize the cholinergic receptor protein. Proc. Natl. Acad. Sci. USA. , 67 , 1241-1247.
3. Brown, A. M. (1991) A cellular logic for G-protein-coupled ion channel path map. FESEB J. 5 , 2175-2179.
4. Lee, Y. C. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms. Ann. Rev. Pharmacol. , 12 , 265-286.
5. Harvey, A. L. and Anderson, A. J. (1991) Dendrotoxins: Snake toxins that block potassium channel and facilitate neurotransmitter release in snake toxins. Pergamon Press, pp. 131-164.
6. Ducancel, F., Rowan, E.G., Cassar, Harvey, A. L., Medez , A and Boulain , J. C. (1991) Amino acid sequence of a muscarinic toxin deduced from the cDNA nucleotide sequence. Toxicon, 29 , 516-520.
7. Adem, A., Asblom, A., Johansson, G. and Karlsson, E. (1988) Toxins from the venom of the green mamba Dendroaspis angustiops. That inhibit the binding of quinuclidinyl benzilate to muscarinic acetylcholine receptor. Biochim. Biophys. Acta. , 968 , 340-345.
8. Rossenberg, P. (1988) Phospholipase A2 Toxins. in Snake Toxins in Neurochemistry. Dolly, J.O. ed. Halsted Press, New York, USA, pp. 27-46.
9. Harvey, A. L., Marshall, R. J. and Karlsson, E. (1982) Effects of purified cardiotoxins from the Thailand cobra (Naja naja siamensis) on isolated skeletal and cardiac muscle preparations. Toxicon, 20 , 379-396.
10. Hodges, S. J., Agbaji, A. S., Harvey, A. L.,and Hider, R. C. (1987) Cobra cardiotoxins: Purification,effects on skeletal muscle and structure/activity relationships. Eur. J. Biochem. , 165 , 373-383.
11. Chang, C. C., Chung, S. T., Lee, C. Y. and Wei, J. W. (1972) Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom. Br. J. Pharmac. , 44 , 752-764.
12. Teng, C. M.,Jy. W and Ouyang, C. (1984) Cardiotoxin from Naja naja atra snake venom: A potentiator of platelet affregation. Toxicon , 22 , 463-470.
13. Jiang, X. L. and Hinman, C. L. (1990) Ablation of natural killer cell function by solution cardiotoxin. Int. J. Immunopharmac. , 12 , 247-254.
14. Kini, R. M., Har, N. C. and Evans, H. J. (1988) Non-enzymatic inhibitors of coagulation and platelet aggregation from Naja nigricollis venom are cardiotoxins. Biochem. Biophys. Res. Commun., 150 , 1012-1016.
15. Bougis, P. E., Khelif, A. A. and Rochat, H. (1989) On the inhibition of [Na+, K+]-ATPase by the components of Naja mossambica mossambica venom: Evidence for two distinct rat brain [Na+, K+]-ATPase activities. Biochemistry , 28 , 3037-3043.
16. Lee, C. Y., Lin, J. S. and Wei, J. W. (1970) Identification of cardiotoxin with Cobra mine B, DLF, Toxin γ and cobra venom cytotoxin. 2nd Intern. Symp. Animal and Plant Toxins, Tel-Aviv Israel, pp. 307-318.
17. Braganca, B. M., Patel, N. T. and Badrinath, P. G. (1967) Isolation and properties of a cobra venom factor selectively cytotoxic to Yoshida sarcoma cells., Biochim. Biophys. Acta. , 136 , 508-520.
18. Arms, K. and Mcpheeters, D. (1975) Sensitivity of cultured embryonic heart cells to cardiotoxin obtained from Naja naja siamensis venom. Toxicon, 13 , 333-338.
19. Tzeng, W. F. and Chen, Y. H. (1988) Suppression of snake-venom cardiotoxin-induced cardiomyocyte degradation by blockage of Ca2+ influx or inhibition of nonlysosomal proteinases., Biochem. J., 256 , 89-95.
20. B. Rees, A. Bilwes, J. P. Samama and D. Moras (1990) Cardiotoxin VII4 from Naja mossambica mossambica the refined crystal structure J. Mol. Biol., 214 , 281-297.
21. Steinmetz, W. E., Bougis, P. E., Rochat,H., Braun, W. and Wuthrich,K. (1988) 1H nuclear-magnetic resonance studies of the three-dimensional structure of the cardiotoxin CTX Iib from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins. Eur. J. Biochem., 172 , 101-116.
22. Bilwes, A., Rees, B. and D. Moras (1994) X-ray structure at 1.55 Å of Toxin γ , a Cardiotoxin from Naja nigricollis Venom. J. Mol. Biol., 239 , 122-136.
23. Singhal, A. K., Chien, K. Y., Wu, W. G. and Gordon S. Rule (1993) Solution structure of Cardiotoxin V from Naja naja atra. Biochemistry, 32 , 8036-8044.
24. Sun, Y. J., Wu, W. G., Chiang, C. M., Hsin, A. Y. and Hsiao, C. D. (1997) Crystal structure of Cardiotoxin V from Taiwan Cobra Venom: pH-Dependent conformational change and a novel membrane-binding motif identified in the Three-Finger Loops of P-type Cardiotoxin. Biochemistry, 36 , 2403-2413.
25. Wong, C. H., Chen, Y. H., Hung, M. C., Wang, K. T., Ho, C. L. and Lo, T. B. (1978) Renaturation of a reduced Taiwan cobra cardiotoxin. Biochim. Biophys. Acta, 533 , 105-111.
26. Hung, M. C., Pan, Y. H., Cheng, K. L. and Chen, Y. H. (1978) The status of tyrosyl residues in a Formosan cobra cardiotoxin. Biochim. Biophys. Acta, 535 , 178-187.
27. Carlsson, F. H. H. (1980) The preparation of nitrotyrosyl derivatives of three elapid venom cardiotoxins., Biochim. Biophys. Acta. , 624 , 460-472.
28. Gatineau, E., Toma, F., Montenay-Garestier, T., Takechi, M. and Menez, A. (1987) Role of tyrosine and tryptophan residues in the structure-activity relationships of a cardiotoxin from Naja nigricollis venom. Biochemistry, 26 , 8046-8055.
29. Kini, M. R. and Evans, H. J. (1989) Role of cationic residues in cytolytic activity: Modification of lysine residues in the cardiotoxin from Naja nigricollis venom and correlation between cytolytic and antiplatelet activity. Biochemistry, 28 , 9209-9215.
30. Gatineau, E., Takechi, M., Bouet, F., Mansuelle, P., Rochat, H., Harvey, A. L., Montenay-Garestier, Th. And Menez, A. (1990) Delineation of the functional site of a snake venom cardiotoxin: Preparation, structure, and function of monoacetylated derivatives. Biochemistry, 29 , 6480-6489.
31. Carlsson, F. H. H. (1987) The selective S-alkylation of a methionine residue in an elapid venom cardiotoxin. Int. J. Biochem., 19 , 915-921.
32. Carlsson, F. H. H. and Louw, A. I. (1978) The oxidation of methionine and its effect on the properties of cardiotoxin VII from naja melanoleuca venom. Biochim. Biophys. Acta , 534 , 322-330.
33. Dubovskii, P. V., Dementieva, D. V., Bocharov, E. V., Utkin, Y. N. and Arseniev, A. S. (2001) Membrane binding motif of the P-type Cardiotoxin. J. Mol. Biol., 305 , 137-149.
34. Chang, L. S., Huang, H. B. and Lin, S. R. (2000) The multiplicity of cardiotoxins from Naja naja atra (Taiwan cobra) venom., Toxicon , 38 , 1065-1076.
35. Lin, S. R., Chang, L. S. and Chang, K. L. (2002) Separation and structure-function studies of Taiwan cobra cardiotoxins. Journal of Protein Chemistry , 21 , 81-86.
36. Yang, C. C., King, K. and Sun, T. P. (1981) Chemical modification of lysine and histidine residues in phospholipase A2 from venom of Naja naja atra (Taiwan cobra). Toxicon, 19 ,645-659.
37. Lo, T. B., Chen Y. H. and Lee, C. Y. (1966) Chemical studies of Formosan cobra (Naja naja atra) venom. Part I. Chromatographic separation of crude venom on CM-Sephadex and prelimilary characterization of its components. J Chinese Chem. Soc. Ser II, 13 , 25-37.
38. Joubert, F. J. (1977) Naja mossambica mossambica venom: purification, some properties and the amino acid sequence of the three phospholipase A (CM-I , CM-II and CM-III). Biochim. Biophys. Acta. , 493 , 216-227.
39. Otwinowski, Z. and Minor, W. (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode Methods in Enzymology., Macromolecular Crystallography, part A , 276 , p.307-326.
40. Blundell, T. L. and Johnson, L. N. (1976) in Protein Crystallography, Academic Press.
41. Eisenberg, D. and Hill, C. P. (1989) Protein crystallography : more surprises ahead. Trends Biochem. Sci., 14 , 260-264.
42. Gluster, J. P. and Trueblood, K. N. (1985) in Crystal Structure Analysis : A Primer, Oxford University Press.
43. Ladd, M. F. C. and Palmer, R. A. (1985) in Structure determination by X-ray crystallography. Plenum press, New York.
44. Mcpherson, A. (1982) in The preparation and analysis of protein crystals. , John Wiley & Sons.
45. Rao, S. N., Jih, J. H. and Hartsuck, J. A. (1980) Rotation-function and space groups. Acta cryst. , A36 , 878-884.
46. Tollin, P., Main, P. and Rossmann, M. G. (1966) The symmetry of the rotation function., Acta Cryst. , 20 ,404-407.
47. Crowther, R. A. and Blow, D. M. (1967) A method of positioning a known molecule in an unknown crystal structure. Acta. Cryst. , 23 , 544-548.
48. Brunger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., P. Gros, J. M., Pannu, N. S., Read, R. J., Rice, L. M. (1998) Crystallography and NMR System (CNS)：A new software system for macromolecular structure determination. Acta. Cryst. , D54 , 905-921.
49. DeLano, W. L. and Brunger, A. T. (1995) The Direct Rotation Function: Rotational Patterson Correlation Search Applied to Molecular Replacement, Acta Cryst. , D 51 , 740-748.
50. Tong, L. and Rossmann, M. G.(1988) Rotation function calculations with GLRF program. Meth. Enzymo. , 276 , 594-611.
51. Huber, R., Die Automatisierte Faltmolekuelmethode. (1965) Acta Cryst. , A19 , 353-356.
52. Brunger, A. T. (1990) Extension of molecular replacement: A new search strategy based on Patterson correlation refinement, Acta Cryst. , A46 , 46-57.
53. Read, R. J. and Schierbeek, A. J. J. (1988) A phased translation function, Appl. Cryst. , 21 , 490-495.
54. Fujinaga, M. and Read, R. J. (1987) Experiences with a new translation function program. J. Appl. Crystallogr. , 20 , 517-521.
55. Navaza, J. and Vernoslova, E. (1995) On the fast translation function for molecular replacement, Acta Cryst. , A51 , 445-449.
56. Brunger, A. T. (1992) The Free R Value: a Novel Statistical Quantity for Assessing the Accuracy of Crystal Structures, Nature , 355, 472-474.
57. Adams, P. D., Pannu, N. S., Read, R. J. and Brunger, A. T. (1997) Cross -validated Maximum Likelihood Enhances Crystallographic Simulated Annealing Refinement., Proc. Natl. Acad. Sci. USA 94, 5018-5023.
58. Pannu, N. S. and Read, R. J. (1996) Improved structure refinement through maximum likelihood., Acta Cryst. A52 , 659-668.
59. Brunger, A. T., Kuriyan, J. and Karplus, M. (1987) Crystallographic R factor Refinement by Molecular Dynamics., Science, 235, 458-460.
60. Brunger, A. T., Krukowski, A. and Erickson, J. (1990) Slow-Cooling Protocols for Crystallographic Refinement by Simulated Annealing, Acta Cryst., A46, 585-593.
61. Rice, L. M. and Brunger, A. T. (1994) Torsion Angle Dynamics: Reduced Variable Conformational Sampling Enhances Crystallographic Structure Refinement, Proteins: Structure, Function, and Genetics, 19, 277-290.
62. Pannu, N. S. and Read, R. J. (1996) Improved structure refinement through maximum likelihood., Acta Cryst. A52, 659-668.
63. Adams, P. D., Pannu, N. S., Read, R. J. and Brunger, A. T. (1997) Cross-validated Maximum Likelihood Enhances Crystallographic Simulated Annealing Refinement., Proc. Natl. Acad. Sci., USA 94, 5018-5023.
64. Read, R. J. (1986) Improved Fourier coefficients for maps using phases from partial structures with errors., Acta Cryst., A42 , 140-149.
65. Bhat, T. N. (1988) Calculation of an OMIT map. J Appl Crystallogr 21, 279-281.
66. Hodel, A., Kim, S. H. and Brunger, A. T. (1992) Model Bias in Macromolecular Crystal Structures., Acta Cryst., A48 , 851-859.
67. Rice, L. M. and Brunger, A. T. (1994) Torsion Angle Dynamics: Reduced Variable Conformational Sampnameling Enhances Crystallographic Structure Refinement, Proteins: Structure, Function, and Genetics, 19 , 277-290.
68. Brunger, A. T., Adams, P. D. and Rice, L. M. (1997) New applications of simulated annealing in X-ray crystallography and solution NMR, Structure 5 , 325-336.
69. Kleywegt, G. J. and Brunger, A. T. (1996) Checking your imagination: Applications of the free R value. , Structure 4, 897-904.
70. Bernard Gilquin, Christian Roumestand, Sophie Zinn-Justin, Andre Menez, and Flavio Toma (1993) Refined three-dimensional solution structure of a snake cardiotoxin : Analysis of the side-chain organization suggests the existence of a possible phospholipid binding site. Biopolymers , 33 , 1659-1675.
71. Sue, S. C., Jarrell, H. C., Brisson, J. R. and Wu, W. G. (2001) Dynamic characterization of the water binding loop in the P-type Cardiotoxin : Implication for the role of the bound water molecule. Biochemistry , 40 , 12782-12794.
72. Dementieva, D. V., Bocharov, E. V. and Arseniev, A. S. (1999) Two forms of cytotoxin II (cardiotoxin) from Naja naja oxiana Spatial structures with tightly bound water molecules. Eur. J. Biochem. 263 , 152-162.
73. Dement’eva, D. V., Utkin, Yu. N. and Arseniev, A. S. (1996) Secondary structure and conformational heterogeneity of cytotoxin II from Naja naja oxiana. Russian Journal of Bioorganic Chemistry , 22 , 289-302.