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NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1.

ABSTRACT: Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake ?-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 ?M, ws-LYNX1 competed with (125)I-?-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing ?7 nAChRs to 1 ?M ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on ?4?2 and ?3?2 nAChRs. Increasing ws-LYNX1 concentration to 10 ?M caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.

SUBMITTER: Lyukmanova EN

PROVIDER: S-EPMC3060513 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1.

Lyukmanova Ekaterina N EN Shenkarev Zakhar O ZO Shulepko Mikhail A MA Mineev Konstantin S KS D'Hoedt Dieter D Kasheverov Igor E IE Filkin Sergey Yu SY Krivolapova Alexandra P AP Janickova Helena H Dolezal Vladimir V Dolgikh Dmitry A DA Arseniev Alexander S AS Bertrand Daniel D Tsetlin Victor I VI Kirpichnikov Mikhail P MP

The Journal of biological chemistry 20110120 12

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking ...[more]

PMID: 21252236

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Project description:Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the ?7 nAChR extracellular ligand-binding domain and the transmembrane domain of ?1 glycine receptor (?7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at ?7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on ?7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for ?-bungarotoxin and similar snake ?-neurotoxins also targeting ?7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

Dataset Information

NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1.

Publications

NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1.

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