Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A₂

Abstract

The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A2 (PLA2s) is not yet fully elucidated. Recently, new high‐affinity binding proteins for PLA2 toxins have been discovered, including the important intracellular Ca2+ sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA2s with the Ca2+‐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 PLA2 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 PLA2 toxin.

Protein Engineering in Structure-Function Studies of Viper's Venom Secreted Phospholipases A2

“…Our structure-function studies of ß-neurotoxic sPLA 2 s (Ivanovski et al, 2000(Ivanovski et al, , 2004Petan et al, 2002Petan et al, , 2005Prijatelj et al, 2000Prijatelj et al, , 2002Prijatelj et al, , 2003Prijatelj et al, , 2006bPrijatelj et al, , 2008Pungerčar et al, 1999) clearly show that different parts of the toxin molecule have separate roles in the distinct steps of the complex mechanism of presynaptic neurotoxicity . Most significantly, by selectively mutating parts of the DPLA 2 molecule, we were able to map the residues that separate the weakly ß-neurotoxic sPLA 2 from the 150-fold more potent AtxA (Prijatelj et al, 2008).…”

“…However, the latter is almost 150-fold more toxic in mice (Prijatelj et al, 2003). To our great surprise, the introduction of the YIRN cluster into DPLA 2 did not increase its toxicity; on the contrary, the DPLA 2 YIRN mutant was more than five times less toxic than DPLA 2 (Prijatelj et al, 2003). Additionally, our study on the importance of the N-terminal residue Phe24, in which it was replaced by other aromatic (tyr or trp), polar uncharged (ser or asn) or hydrophobic (ala) residues, suggested that Phe24 is also involved in the neurotoxicity of Atxs, apparently at a stage not involving enzymatic activity or interactions with the high-affinity binding proteins R25, R180 and CaM (Petan et al, 2002).…”

“…Thus, the lethal potency of the AtxA-Y115K/I116K/R118M/N119L (AtxA KKML ) mutant was 290-fold lower than that of AtxA (Ivanovski et al, 2000 and The KKML cluster is present in the weakly neurotoxic DPLA 2 from the venom of Russell's viper, Daboia (Vipera) russelii russelii, which shares a high level of amino acid identity (82%) with AtxA. However, the latter is almost 150-fold more toxic in mice (Prijatelj et al, 2003). To our great surprise, the introduction of the YIRN cluster into DPLA 2 did not increase its toxicity; on the contrary, the DPLA 2 YIRN mutant was more than five times less toxic than DPLA 2 (Prijatelj et al, 2003).…”

“…While Atxs and hGIIA sPLA 2 do not contain a Trp on their IBS, DPLA 2 , a weakly neurotoxic sPLA 2 from Russell's viper that differs from AtxA in only 22 residues, has a Trp residue at position 31 and yet displays interfacial binding and kinetic properties similar to those of AtxA (Petan et al, 2005;Prijatelj et al, 2003Prijatelj et al, , 2008. Besides V31W, DPLA 2 contains several other substitutions of equivalent AtxA IBS residues: L19I, T20P, F24S, S67N, T70S, R118M and N119L (Petan et al, 2005;Prijatelj et al, 2008).…”

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA2 (F6a) from Crotalus durissus collilineatus Snake Venom

“…This region probably is involved in the neurotoxic activity of this PLA 2 . Several studies made with other PLA 2 also showed that C-terminal region play important role for the neurotoxic effect and the difference in the amino acid sequences of this region has a evident relationship to neurotoxic potency (Krizaj, 1989;Curin-Serbec, 1991;Lomonte, 2003;Prijatelj, 2003). Thus these conserved basic amino acid residues seem to be involved in the neurotoxic effect of new crotoxin B isoform PLA 2 F6a from the Crotalus durissus collilineatus but it is not the unique region responsible for this effect.…”