Bacterial expression, NMR, and electrophysiology analysis of chimeric short/long-chain alpha-neurotoxins acting on neuronal nicotinic receptors.
Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.
Lyukmanova
Ekaterina N
EN
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Str., 117997 Moscow, Russia.
Shenkarev
Zakhar O
ZO
Schulga
Alexey A
AA
Ermolyuk
Yaroslav S
YS
Mordvintsev
Dmitry Yu
DY
Utkin
Yurii N
YN
Shoulepko
Mikhail A
MA
Hogg
Ron C
RC
Bertrand
Daniel
D
Dolgikh
Dmitry A
DA
Tsetlin
Victor I
VI
Kirpichnikov
Mikhail P
MP
D000595:Amino Acid Sequence; D000818:Animals; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
D009498:Neurotoxins; D011978:Receptors, Nicotinic