Abstract
Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, γ-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR α-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined ‘loops’ A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer’s disease and nicotine addiction.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ortells, M. O. & Lunt, G. G. Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci. 18, 121–127 (1995).
Corringer, P. J., Le Novère, N. & Changeux, J. P. Nicotinic receptors at the amino-acid level. Annu. Rev. Pharmacol. Toxicol. 40, 431–458 (2000).
Changeux, J. P. & Edelstein, S. J. Allosteric receptors after 30 years. Neuron 21, 959–980 (1998).
Arias, H. R. Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors. Neurochem. Int. 36, 595–645 (2000).
Paterson, D. & Nordberg, A. Neuronal nicotinic receptors in the human brain. Prog. Neurobiol. 61, 75–111 (2000).
Galzi, J. L. et al. Identification of a novel amino acid alpha-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites. J. Biol. Chem. 265, 10430–10437 (1990).
Dennis, M. et al. Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site. Biochemistry 27, 2346–2357 (1988).
Kao, P. N. & Karlin, A. Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues. J. Biol. Chem. 261, 8085–8088 (1986).
Middleton, R. E. & Cohen, J. B. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label. Biochemistry 30, 6987–6997 (1991).
Fu, D. X. & Sine, S. M. Competitive antagonists bridge the alpha-gamma subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions. J. Biol. Chem. 269, 26152–26157 (1994).
O’Leary, M. E., Filatov, G. N. & White, M. M. Characterization of d-tubocurarine binding site of Torpedo acetylcholine receptor. Am. J. Physiol. 266, C648–653 (1994).
Corringer, P. J. et al. Identification of a new component of the agonist binding site of the nicotinic alpha 7 homo-oligomeric receptor. J. Biol. Chem. 270, 11749–11752 (1995).
Sine, S. M., Kreienkamp, H. J., Bren, N., Maeda, R. & Taylor, P. Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity. Neuron 15, 205–211 (1995).
Czajkowski, C., Kaufmann, C. & Karlin, A. Negatively charged amino-acid residues in the nicotinic receptor delta subunit that contribute to the binding of acetylcholine. Proc. Natl Acad. Sci. USA 90, 6285–6289 (1993).
Martin, M., Czajkowski, C. & Karlin, A. The contributions of aspartyl residues in the acetylcholine receptor gamma and delta subunits to the binding of agonists and competitive antagonists. J. Biol. Chem. 271, 13497–13503 (1996).
Prince, R. J. & Sine, S. M. Molecular dissection of subunit interfaces in the acetylcholine receptor. Identification of residues that determine agonist selectivity. J. Biol. Chem. 271, 25770–25777 (1996).
Dougherty, D. A. Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science 271, 163–168 (1996).
Unwin, N. Nicotinic acetylcholine receptor at 9 Å resolution. J. Mol. Biol. 229, 1101–1124 (1993).
Unwin, N. Acetylcholine receptor channel imaged in the open state. Nature 373, 37–43 (1995).
Beroukhim, R. & Unwin, N. Three-dimensional location of the main immunogenic region of the acetylcholine receptor. Neuron 15, 323–331 (1995).
Miyazawa, A., Fujiyoshi, Y., Stowell, M. & Unwin, N. Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel wall. J. Mol. Biol. 288, 765–786 (1999).
West, A. P. Jr, Bjorkman, P. J., Dougherty, D. A. & Lester, H. A. Expression and circular dichroism studies of the extracellular domain of the alpha subunit of the nicotinic acetylcholine receptor. J. Biol. Chem. 272, 25468–25473 (1997).
Schrattenholz, A. et al. Expression and renaturation of the N-terminal extracellular domain of Torpedo nicotinic acetylcholine receptor alpha-subunit. J. Biol. Chem. 273, 32393–32399 (1998).
Alexeev, T. et al. Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli. Eur. J. Biochem. 259, 310–319 (1999).
Wells, G. B., Anand, R., Wang, F. & Lindstrom, J. Water-soluble nicotinic acetylcholine receptor formed by alpha7 subunit extracellular domains. J. Biol. Chem. 273, 964–973 (1998).
Tierney, M. L. & Unwin, N. Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor. J. Mol. Biol. 303, 185–196 (2000).
Smit, A. B. et al. A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature 411, 261–268 (2001).
Bork, P., Holm, L. & Sander, C. The immunoglobulin fold. Structural classification, sequence patterns and common core. J. Mol. Biol. 242, 309–320 (1994).
Le Novère, N., Corringer, P. J. & Changeux, J. P. Improved secondary structure predictions for a nicotinic receptor subunit: incorporation of solvent accessibility and experimental data into a two-dimensional representation. Biophys. J. 76, 2329–2345 (1999).
Holm, L. & Sander, C. Dali/FSSP classification of three-dimensional protein folds. Nucleic Acids Res. 25, 231–234 (1997).
Tzartos, S. J. et al. The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies. Mol. Neurobiol. 5, 1–29 (1991).
Fernando Valenzuela, C., Weign, P., Yguerabide, J. & Johnson, D. A. Transverse distance between the membrane and the agonist binding sites on the Torpedo acetylcholine receptor: a fluorescence study. Biophys. J. 66, 674–682 (1994).
Galzi, J. L., Bertrand, S., Corringer, P. J., Changeux, J. P. & Bertrand, D. Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor. EMBO J. 15, 5824–5832 (1996).
Machold, J., Weise, C., Utkin, Y., Tsetlin, V. & Hucho, F. The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica. Eur. J. Biochem. 234, 427–430 (1995).
Zhong, W. et al. From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor. Proc. Natl Acad. Sci. USA 95, 12088–12093 (1998).
Harel, M. et al. Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase. Proc. Natl Acad. Sci. USA 90, 9031–9035 (1993).
Hemmingsen, J. M., Gernert, K. M., Richardson, J. S. & Richardson, D. C. The tyrosine corner: a feature of most Greek key beta-barrel proteins. Protein Sci. 3, 1927–1937 (1994).
Mishina, M. et al. Location of functional regions of acetylcholine receptor α-subunit by site-directed mutagenesis. Nature 313, 364–369 (1985).
Green, W. N. & Wanamaker, C. P. Formation of the nicotinic acetylcholine receptor binding sites. J. Neurosci. 18, 5555–5564 (1998).
Eisele, J. L. et al. Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature 366, 479–483 (1993).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Leslie, A. G. Integration of macromolecular diffraction data. Acta Crystallogr. D Biol. Crystallogr. 55, 1696–1702 (1999).
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).
La Fortelle, E. de & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement in the MIR and MAD methods. Methods Enzymol. 276, 472–494 (1997).
Kleywegt, G. J. & Jones, T. A. in From First Map to Final Model (eds Bailey, S., Hubbard, R. & Waller, D.) 59–66 (SERC Daresbury Laboratory, Warrington, 1994).
Cowtan, K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38 (1994).
Jones, T. A., Zou, J-Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).
Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 (1997).
Acknowledgements
We thank R. van der Schors for mass spectrometry; R. van Elk for HEPES binding assays; A. Perrakis and U. Gohlke for helpful suggestions; M. Lamers for help with figure preparation; and beam line scientists at ESRF and EMBL outstations Hamburg and Grenoble, in particular W. Rypniewski and G. Leonard for assistance during data collection. Nederlandse Organisatie voor Wetenschappelijk Onderzoek-Chemische Wetenschappen and Stichting voor de Technische Wetenschappen are acknowledged for financial support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Brejc, K., van Dijk, W., Klaassen, R. et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269–276 (2001). https://doi.org/10.1038/35077011
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/35077011
This article is cited by
-
The modes of action of ion-channel-targeting neurotoxic insecticides: lessons from structural biology
Nature Structural & Molecular Biology (2023)
-
In vitro culture using nicotine and d-tubocurarine and in silico analysis depict the presence of acetylcholine receptor (AChR) in tomato (Solanum lycopersicum L.)
In Vitro Cellular & Developmental Biology - Plant (2023)
-
Structure-function Studies of GABA (A) Receptors and Related computer-aided Studies
Journal of Molecular Neuroscience (2023)
-
Origin of acetylcholine antagonism in ELIC, a bacterial pentameric ligand-gated ion channel
Communications Biology (2022)
-
Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide
Scientific Reports (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.