Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A sub(2)

The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A sub(2) (PLA sub(2)s) is not yet fully elucidated. Recently, new high-affinity binding proteins for PLA sub(2) toxins have been discovered, including the important intracellular Ca super(2+) sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA sub(2)s with the Ca super(2+)-bound form of CaM was investigated by mutational analysis of ammodytoxin A (AtxA) from the long-nosed viper (Vipera ammodytes ammodytes). Several residues in the C-terminal part of AtxA were found to be important in this interaction, particularly those in the region 115-119. In support of this finding, introduction of Y115, I116, R118 and N119, present in AtxA, into a weakly neurotoxic PLA sub(2) from Russell's viper (Daboia russellii russellii) increased by sevenfold its binding affinity for CaM. Furthermore, two out of four peptides deduced from different regions of AtxA were able to compete with the toxin in binding to CaM. The nonapeptide showing the strongest inhibition was that comprising the AtxA region 115-119. This stretch contributes to a distinct hydrophobic patch within the region 107-125 in the C-terminal part of the molecule. This lacks any substantial helical structure and is surrounded by several basic residues, which may form a novel binding motif for CaM on the molecular surface of the PLA sub(2) toxin.