P05, a new leiurotoxin I-like scorpion toxin: synthesis and structure-activity relationships of the alpha-amidated analog, a ligand of Ca(2+)-activated K+ channels with increased affinity.
The venom of the scorpion Androctonus mauretanicus mauretanicus contains a toxin, P05, which is structurally and functionally similar to scorpion leiurotoxin I (87% sequence identity), a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges. A C-terminal carboxyl-amidated analog of P05 (sP05-NH2) was chemically synthesized by the solid-phase technique and fully characterized. Toxicity assays in vivo established that sP05-NH2, like native P05, is a potent and lethal neurotoxic agent in mice (LD50 of 20 ng per mouse). Pharmacological assays in vitro however showed that, unlike P05 which has a binding affinity of 2 x 10(-11) M, sP05-NH2 apparently binds irreversibly to the apamin receptor. Iodination at the C-terminal His gave diiodo-sP05-NH2, which had a binding affinity similar to that of native P05. The disulfide bridge pairings were chemically determined for sP05-NH2 and thereby deduced for P05 and leiurotoxin I: linkages were between Cys3 and Cys21, Cys8 and Cys26, and Cys12 and Cys28. Molecular dynamics refinement of P05 also using data from leiurotoxin I suggests that P05 is mainly composed of a double-stranded, antiparallel beta-sheet (from Leu18 to Val29) linked to an alpha-helix (from Arg6 to Gly16) by two disulfides (Cys8-Cys26 and Cys12-Cys28) and to an extended fragment (from Thr1 to Leu5) by the third disulfide (Cys3-Cys21). In agreement with the model, circular dichroism analysis of sP05-NH2 showed that the toxin structure is highly rigid.(ABSTRACT TRUNCATED AT 250 WORDS)
Sabatier
J M
JM
Laboratoire de Biochimie, CNRS URA 1455, Faculté de Médecine Nord, Marseille, France.
Zerrouk
H
H
Darbon
H
H
Mabrouk
K
K
Benslimane
A
A
Rochat
H
H
Martin-Eauclaire
M F
MF
Van Rietschoten
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001030:Apamin / Q000378:metabolism; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002942:Circular Dichroism; D007202:Indicators and Reagents; D007700:Kinetics; D007928:Lethal Dose 50; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000138:chemical synthesis* / Q000633:toxicity; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011487:Protein Conformation; D051381:Rats; D012604:Scorpion Venoms / Q000138:chemical synthesis* / Q000494:pharmacology* / Q000633:toxicity; D013329:Structure-Activity Relationship; D013574:Synaptosomes / Q000378:metabolism
D007202:Indicators and Reagents; D009498:Neurotoxins; D015221:Potassium Channels; D012604:Scorpion Venoms; C080397:scorpion venom P05; C080398:scorpion venom P05-NH2; D001030:Apamin; D002118:Calcium