On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding.
We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.
Danse
J M
JM
Département d'Ingénierie et d'Etudes des Protéines (DIEP), CEA, Saclay, Gif-sur-Yvette, France.
Rowan
E G
EG
Gasparini
S
S
Ducancel
F
F
Vatanpour
H
H
Young
L C
LC
Poorheidari
G
G
Lajeunesse
E
E
Drevet
P
P
Ménez
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D002552:Cerebral Ventricles / Q000187:drug effects / Q000473:pathology; D002645:Chickens; D004546:Elapid Venoms / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D005260:Female; D005813:Genes, Synthetic; D005973:Glutamine; D066298:In Vitro Techniques; D007276:Injections, Intraventricular; D007700:Kinetics; D007928:Lethal Dose 50; D008239:Lysine; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D013574:Synaptosomes / Q000378:metabolism
D004546:Elapid Venoms; D009498:Neurotoxins; D015221:Potassium Channels; D011994:Recombinant Proteins; D005973:Glutamine; C020273:dendrotoxin; D008239:Lysine