Role of tyrosine and tryptophan residues in the structure-activity relationships of a cardiotoxin from Naja nigricollis venom.

This paper is an attempt to localize the critical area determining toxicity in a snake cardiotoxin. Toxin gamma is a single-chain polypeptide of 60 amino acids, which has been isolated from the venom of the African spitting cobra, Naja nigricollis. Three aromatic residues, namely, Trp-11, Tyr-22, and Tyr-51, have been individually modified by chemical means. The structure of the native toxin and of each derivative has been carefully investigated by circular dichroism, fluorescence, proton magnetic resonance spectroscopy, and two specific monoclonal antibodies. None of the chemical modifications alters the overall structure of the toxin, which in all cases remains folded into three adjacent loops (I, II, and III) rich in beta-pleated sheet emerging from a small globular region containing four disulfide bridges. A number of subtle changes, however, have been detected in the structure of each derivative compared with that of the native toxin. In particular, nitration of Tyr-51 provoked a structural perturbation in the globular region. Nitration of Tyr-22 induces a more substantial change in the beta-sheet area of the molecule. Thus, the strong inter-ring NOE that is observed in the native toxin between Tyr-22 and Tyr-51 vanishes in the Tyr-22 derivative, and significant changes are observed in the globular region. In contrast, no alteration of the beta-sheet structure of loops II and III has been detected after modification of Trp-11. All changes observed for this derivative remain located in the vicinity of the indole side chain of Trp-11 in loop I. The biological consequences of the modifications were measured: the lethal potency in vivo in mice and the cytotoxic activities in vitro on FL-cells. Lethal activities correlate with cytotoxicity: Tyr-51 modified toxin is equally potent as native toxin, whereas Tyr-22 and Trp-11 derivatized toxins are characterized by substantially lesser activities, the Trp-11 derivatized toxin being the least potent. We conclude that (1) Tyr-51 is not involved in the functional site of the toxin, although it is in interaction with the core of the molecule, (2) Tyr-22 may play a dual structural and functional role, and (3) Trp-11 is in, or in close proximity to, the functional site of the toxin. These data indicate the importance of loop I in determining toxicity of the cardiotoxin.