Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A2.
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.
Prijatelj
Petra
P
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
Sribar
Jernej
J
Ivanovski
Gabriela
G
Krizaj
Igor
I
Gubensek
Franc
F
Pungercar
Joze
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017840:Daboia; D017386:Sequence Homology, Amino Acid; D014757:Viper Venoms / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D017819:Viperidae
D002147:Calmodulin; D017931:DNA Primers; D010455:Peptides; D011994:Recombinant Proteins; D014757:Viper Venoms; C068117:ammodytoxin A; D010741:Phospholipases A; D054467:Phospholipases A2