Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23.
Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N-hydroxysuccinimide esters of p-azidobenzoic, p-azidotetraflourobenzoic, p-benzoylbenzoic and p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acids. The ion-exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross-linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross-linking yield (up to 16% of the bound toxin) was observed for azidobenzoyl-Lys23-CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group: the azido derivatives labelled the gamma and delta subunits, benzoylbenzoyl derivative labelled the alpha and delta subunits, while p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl derivative reacted with alpha, gamma and delta subunits. The cross-linking experiments in the presence of varying concentrations of (+)-tubocurarine demonstrated that the Lys23-attached diazirinyl group contacts the delta and alpha subunits in one ligand-binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23-diazirinyl are with gamma and alpha subunits. This means that the central loop in the two CTX molecules binds at the alpha/gamma and alpha/delta interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X-ray structure of CTX, showed that this value does not exceed 13 A. The results obtained favor the disposition of the ligand-binding sites at the subunit interfaces, with the distance between alpha and delta, or alpha and gamma subunits at these sites being not more than 13 A.
Utkin
Y N
YN
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow. utkin@ibch.siobc.ras.ru
Krivoshein
A V
AV
Davydov
V L
VL
Kasheverov
I E
IE
Franke
P
P
Maslennikov
I V
IV
Arseniev
A S
AS
Hucho
F
F
Tsetlin
V I
VI
D000818:Animals; D001665:Binding Sites; D003039:Cobra Neurotoxin Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004557:Electric Organ / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D019920:Photoaffinity Labels; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014101:Torpedo
D003039:Cobra Neurotoxin Proteins; D003432:Cross-Linking Reagents; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D008239:Lysine