An aromatic, but not a basic, residue is involved in the toxicity of group-II phospholipase A2 neurotoxins.
Ammodytoxins (Atxs) A, B and C are basic phospholipase A2s from Vipera ammodytes ammodytes snake venom, and they exhibit presynaptic toxicity. The most toxic is AtxA, followed by AtxC, its naturally occurring F124-->I/K128-->E mutant, which is 17 times less toxic. Two mutants of AtxA have been produced in bacteria and characterized. The specific enzymic activity of the K128-->E mutant on mixed phosphatidylcholine/Triton X-100 micelles is similar to that of the wild type. The K108-->N/K111-->N mutant, however, possesses 160% of the wild-type activity. Replacement of the two basic residues by uncharged, polar residues on the opposite side of the protein to the enzyme active site and interfacial adsorption surface results in increased enzymic activity at the water/lipid aggregate interface, due to a redistribution of electrostatic charge. The binding affinity of the double mutant for the specific acceptor in bovine brain was similar to that of AtxA, whereas the affinity of the single mutant was similar to that of AtxC, which was slightly weaker than that of AtxA. Interestingly, the substitution of any of these three basic surface residues did not significantly change the lethal potency of AtxA. Since the single mutant AtxA(K128-->E) is equivalent to the AtxC(I124-->F) mutant, this indicates that the residue at position 124 is important for presynaptic toxicity of Atxs. The more than 10-fold lower toxicity of AtxC, compared with AtxA, is a consequence of the substitution of Phe-124 (aromatic ring) with Ile (aliphatic chain). Exposed aromatic residues in the C-terminal region may also be important for the neurotoxicity of other similar toxins.
Pungercar
J
J
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. joze.pungercar@ijs .si
Krizaj
I
I
Liang
N S
NS
Gubensek
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D054501:Group II Phospholipases A2; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D010649:Phenylalanine; D010741:Phospholipases A / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D054467:Phospholipases A2; D011485:Protein Binding; D011994:Recombinant Proteins / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D013570:Synaptic Membranes / Q000187:drug effects; D018675:Toxicity Tests; D014757:Viper Venoms / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*
D009498:Neurotoxins; D011994:Recombinant Proteins; D014757:Viper Venoms; D010649:Phenylalanine; C068117:ammodytoxin A; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C060161:ammodytoxin C