Comparative structural studies of two natural isoforms of ammodytoxin, phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity☆

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

“…The IBS of Atxs (Figure 2, B) is formed by Leu2, Leu3, Leu19, Thr20, Phe24, Val31, Ser67, Lys69, Thr70, Arg72, Arg118, Asn119, and Phe124 (Petan et al, 2005). The slight structural flexibility observed for the exposed side chains of residues in the IBS (e.g., Phe24 in Figure 2, B) is in keeping with the role of these residues in supporting optimal interactions of the molecule with the dynamic structure of phospholipid aggregates (Saul et al, 2010).…”

“…AtxA is the most lethal; and its protein isoforms, AtxC and AtxB, are 17-and 28-fold less potent, respectively (Thouin et al, 1982). The crystal structures of recombinant AtxA (PDB code 3G8G) and natural AtxC (PDB code 3G8H) demonstrate the absence of significant structural differences between the two toxins (Saul et al, 2010). There is only a minor conformational difference at positions 127 and 128 in the C-terminal region, caused by the charge-reversal substitution of Lys128 for Glu, which does not significantly influence the toxicity (Saul et al, 2010).…”

“…The crystal structures of recombinant AtxA (PDB code 3G8G) and natural AtxC (PDB code 3G8H) demonstrate the absence of significant structural differences between the two toxins (Saul et al, 2010). There is only a minor conformational difference at positions 127 and 128 in the C-terminal region, caused by the charge-reversal substitution of Lys128 for Glu, which does not significantly influence the toxicity (Saul et al, 2010). An illustrative example of the subtle structure-function relationships of sPLA 2 s is the conversion, by a single mutation (F22Y), of the gene for bovine pancreatic group IB sPLA 2 to a gene encoding a molecule able to compete with crotoxin, a ß-neurotoxic sPLA 2 from the South American rattlesnake, Crotalus durissus terrificus, for binding to its 45-kDa neuronal-binding protein.…”

“…The catalytic turnover cycle of sPLA 2 s includes a highly conserved His48-Asp99 dyad and an activated water molecule that acts as a nucleophile during hydrolysis of the sn-2 ester bond of the phospholipid (Scott et al, 1990). The resulting tetrahedral intermediate is stabilized by the Saul et al, 2010). Residues important for neurotoxicity are presented in orange mesh surface and extend from the C-terminal region 115-119 (the YIRN cluster), including the spatially close aromatic Phe124 and Phe24, to the N-terminal helix region with the Met7/Gly11 pair in the "lower right" part of the molecule.…”

“…Hence, AtxA has anticoagulant activity. In addition, the 10-fold lower affinity of AtxC for factor Xa, in comparison with AtxA, is due to the (natural) charge-reversal substitution of Lys128 by Glu, leading to a small local conformational change in the C-terminal end of AtxC, which perturbs the interaction with factor Xa (Saul et al, 2010).…”