Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site-directed mutagenesis and photolabelling experiments.
The positive charge of Lys27 was suppressed by chemical means in two short-chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin alpha from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6-15-fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site-directed mutagenesis. This change provokes a 120-fold decrease in the affinity, which reflects a major alteration of toxin-receptor cognate events. Using toxin-alpha derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor-bound state by photocoupling experiments. The delta chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the alpha chain. The toxin derivative weakly labelled the alpha and gamma chains but not the beta chain. The toxin may therefore interact with subunits other than the alpha chain, at least in the vicinity of Lys27.
Hervé
M
M
Département d'Ingénierie et d'Etudes des Protéines, Centre d'Etudes de Saclay, Gif-sur Yvette, France.
Pillet
L
L
Humbert
P
P
Trémeau
O
O
Ducancel
F
F
Hirth
C
C
Ménez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003039:Cobra Neurotoxin Proteins / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004870:Erabutoxins / Q000235:genetics / Q000378:metabolism*; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D003039:Cobra Neurotoxin Proteins; D003432:Cross-Linking Reagents; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; C038908:erabutoxin A; D004870:Erabutoxins; C025154:alpha-cobratoxin; D008239:Lysine