Dimeric α-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors.
In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in αCT-αCT considerably potentiates inhibition of the α3β2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3β2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3β2 nAChR.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul Miklukho-Maklaya 16/10, Moscow 117997, Russia.
Alexey V
AV
Osipov
Prakash
P
Rucktooa
Igor E
IE
Kasheverov
Sergey Yu
SY
Filkin
Vladislav G
VG
Starkov
Tatyana V
TV
Andreeva
Titia K
TK
Sixma
Daniel
D
Bertrand
Yuri N
YN
Utkin
Victor I
VI
Tsetlin
D000478:Alkylation; D001665:Binding Sites; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
D003039:Cobra Neurotoxin Proteins; D004220:Disulfides; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C419362:nicotinic receptor alpha3beta2; C025154:alpha-cobratoxin