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J. Biol. Chem., Vol. 282, Issue 34, 24784-24791, August 24, 2007

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Bacterial Expression, NMR, and Electrophysiology Analysis of Chimeric Short/Long-chain -Neurotoxins Acting on Neuronal Nicotinic Receptors^*

Ekaterina N. Lyukmanova, Zakhar O. Shenkarev, Alexey A. Schulga, Yaroslav S. Ermolyuk, Dmitry Yu. Mordvintsev, Yurii N. Utkin, Mikhail A. Shoulepko, Ron C. Hogg^¶, Daniel Bertrand^¶, Dmitry A. Dolgikh¹, Victor I. Tsetlin, and Mikhail P. Kirpichnikov

From the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Str., 117997 Moscow, Russia, Moscow State University, 119899 Moscow, Russia, and ^¶Department of Neuroscience, Centre Medical Universitaire, Medical Faculty, 1 Rue Michel Servet, CH-1211 Geneva 4, Switzerland

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain -neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain -neurotoxins block both muscle-type and 7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of - and -neurotoxins is essential for their action on the 7 and 32 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 -neurotoxins and -neurotoxins. The correct folding and structural stability for both chimeras were shown by ¹H and ¹H-¹⁵N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I block7 nAChRs with similar potency (IC₅₀ 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain -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 7 nAChR (IC₅₀ 126 nM) but did not convey activity at 32 nAChRs. Docking of both chimeras to7 and32 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of -neurotoxins underlie their selectivity for 32 nAChRs.

Received for publication, December 7, 2006 , and in revised form, May 15, 2007.

^* This work was supported by the Russian Academy of Sciences (program on molecular and cellular biology) and the Russian Foundation for Basic Research (Projects 05-04-48918, 05-04-48932). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 7-495-3368011; Fax: 7-495-3352888; E-mail: dolgikh{at}nmr.ru.

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