Binding of Nucleotide Triphosphates to Cardiotoxin Analogue II from the Taiwan Cobra Venom (Naja naja atra)

Abstract

Snake venom cardiotoxins have been recently shown to block the enzymatic activity of phospholipid protein kinase and Na ؉ ,K ؉ -ATPase. To understand the molecular basis for the inhibitory effects of cardiotoxin on the action of these enzymes, the nucleotide triphosphate binding ability of cardiotoxin analogue II (CTX II) from the Taiwan cobra (Naja naja atra) venom is investigated using a variety of spectroscopic techniques such as fluorescence, circular dichroism, and two-dimensional NMR. CTX II is found to bind to all the four nucleotide triphosphates (ATP, UTP, GTP, and CTP) with similar affinity. Detailed studies of the binding of dATP to CTX II indicated that the toxin molecule is significantly stabilized in the presence of the nucleotide. Molecular modeling, based on the NOEs observed for the dATP⅐CTX II complex, reveals that dATP binds to the CTX II molecule at the groove enclosed between the Nand C-terminal ends of the toxin molecule. Based on the results obtained in the present study, a molecular mechanism to account for the inhibition of the enzymatic activity of the phospholipid-sensitive protein kinase and Na ؉ ,K ؉ -ATPase is also proposed.Snake venom cardiotoxins are small molecular mass (ϳ7 kDa), highly basic proteins cross-linked by four disulfide bridges (1-3). These toxins exhibit a wide variety of biological activities such as contraction of cardiac muscle cells, lysis of erythrocytes, and selective toxicity to certain types of tumor cells (4, 5). More recently, cardiotoxins have been demonstrated to selectively inhibit the action of certain key enzymes such as Na ϩ ,K ϩ -ATPase and phospholipid-sensitive protein kinase (2, 6 -8). Although the general molecular mechanism underlying the enzyme inhibitory action of cardiotoxins is still an enigma, it is contemplated that cardiotoxin could effectively block the enzymatic activities of Na ϩ ,K ϩ -ATPase and phospholipid-sensitive protein kinase by competitively binding (in the present study) to adenosine triphosphate (ATP), which is a key substrate for the functioning of these enzymes. However, to date, the evidence for the binding of a nucleotide triphosphate to cardiotoxin has not been reported.In the present study, for the first time, we demonstrate that the cardiotoxin analogue II (CTX II) 1 isolated from the Taiwan cobra venom (Naja naja atra) bind to nucleotide triphosphates. Herein, we also propose a molecular model of the CTX II⅐dATP complex and suggest a reasonable molecular mechanism to explain the reported inhibitory action of snake venom cardiotoxin on phospholipid-sensitive protein kinase and Na ϩ ,K ϩ -ATPase. MATERIALS AND METHODSCTX II was purified as described by Yang et al. (9). The concentration of the protein was estimated from its absorbance at 280 nm (⑀ 1 mM ϭ 4.08). The nucleotides were purchased from Sigma. The concentration of the nucleotides was estimated (10) from their extinction coefficient values (⑀ 259 ϭ 15,400 for AMP, ADP, ATP, and dATP; ⑀ 271 ϭ 9,000 for CTP; ⑀ 253 ϭ 13,700 for GTP; and ⑀ 260 ϭ 1,000 for UTP). Al...

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