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. 1997 May;6(5):983–998. doi: 10.1002/pro.5560060504

Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

J E Gready 1, S Ranganathan 1, P R Schofield 1, Y Matsuo 1, K Nishikawa 1
PMCID: PMC2143702  PMID: 9144769

Abstract

Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested.

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Selected References

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  1. Akabas M. H., Karlin A. Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit. Biochemistry. 1995 Oct 3;34(39):12496–12500. doi: 10.1021/bi00039a002. [DOI] [PubMed] [Google Scholar]
  2. Banner D. W., D'Arcy A., Janes W., Gentz R., Schoenfeld H. J., Broger C., Loetscher H., Lesslauer W. Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell. 1993 May 7;73(3):431–445. doi: 10.1016/0092-8674(93)90132-a. [DOI] [PubMed] [Google Scholar]
  3. Barnard E. A. Receptor classes and the transmitter-gated ion channels. Trends Biochem Sci. 1992 Oct;17(10):368–374. doi: 10.1016/0968-0004(92)90002-q. [DOI] [PubMed] [Google Scholar]
  4. Benner S. A. Patterns of divergence in homologous proteins as indicators of tertiary and quaternary structure. Adv Enzyme Regul. 1989;28:219–236. doi: 10.1016/0065-2571(89)90073-3. [DOI] [PubMed] [Google Scholar]
  5. Bowie J. U., Lüthy R., Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional structure. Science. 1991 Jul 12;253(5016):164–170. doi: 10.1126/science.1853201. [DOI] [PubMed] [Google Scholar]
  6. Brocchieri L., Karlin S. Geometry of interplanar residue contacts in protein structures. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9297–9301. doi: 10.1073/pnas.91.20.9297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cully D. F., Vassilatis D. K., Liu K. K., Paress P. S., Van der Ploeg L. H., Schaeffer J. M., Arena J. P. Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature. 1994 Oct 20;371(6499):707–711. doi: 10.1038/371707a0. [DOI] [PubMed] [Google Scholar]
  8. Czajkowski C., Karlin A. Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide. J Biol Chem. 1991 Nov 25;266(33):22603–22612. [PubMed] [Google Scholar]
  9. Czajkowski C., Karlin A. Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the delta subunit within 0.9 nm of the 5 alpha subunit-binding. J Biol Chem. 1995 Feb 17;270(7):3160–3164. doi: 10.1074/jbc.270.7.3160. [DOI] [PubMed] [Google Scholar]
  10. Devillers-Thiéry A., Galzi J. L., Eiselé J. L., Bertrand S., Bertrand D., Changeux J. P. Functional architecture of the nicotinic acetylcholine receptor: a prototype of ligand-gated ion channels. J Membr Biol. 1993 Nov;136(2):97–112. doi: 10.1007/BF02505755. [DOI] [PubMed] [Google Scholar]
  11. Edwards Y. J., Perkins S. J. The protein fold of the von Willebrand factor type A domain is predicted to be similar to the open twisted beta-sheet flanked by alpha-helices found in human ras-p21. FEBS Lett. 1995 Jan 30;358(3):283–286. doi: 10.1016/0014-5793(94)01447-9. [DOI] [PubMed] [Google Scholar]
  12. Eiselé J. L., Bertrand S., Galzi J. L., Devillers-Thiéry A., Changeux J. P., Bertrand D. Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities. Nature. 1993 Dec 2;366(6454):479–483. doi: 10.1038/366479a0. [DOI] [PubMed] [Google Scholar]
  13. Falzone C. J., Kao Y. H., Zhao J., Bryant D. A., Lecomte J. T. Three-dimensional solution structure of PsaE from the cyanobacterium Synechococcus sp. strain PCC 7002, a photosystem I protein that shows structural homology with SH3 domains. Biochemistry. 1994 May 24;33(20):6052–6062. doi: 10.1021/bi00186a004. [DOI] [PubMed] [Google Scholar]
  14. García-Guzmán M., Sala F., Sala S., Campos-Caro A., Criado M. Role of two acetylcholine receptor subunit domains in homomer formation and intersubunit recognition, as revealed by alpha 3 and alpha 7 subunit chimeras. Biochemistry. 1994 Dec 20;33(50):15198–15203. doi: 10.1021/bi00254a031. [DOI] [PubMed] [Google Scholar]
  15. Gu Y., Camacho P., Gardner P., Hall Z. W. Identification of two amino acid residues in the epsilon subunit that promote mammalian muscle acetylcholine receptor assembly in COS cells. Neuron. 1991 Jun;6(6):879–887. doi: 10.1016/0896-6273(91)90228-r. [DOI] [PubMed] [Google Scholar]
  16. Herz J. M., Johnson D. A., Taylor P. Distance between the agonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor. J Biol Chem. 1989 Jul 25;264(21):12439–12448. [PubMed] [Google Scholar]
  17. Hubbard S. J., Campbell S. F., Thornton J. M. Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors. J Mol Biol. 1991 Jul 20;220(2):507–530. doi: 10.1016/0022-2836(91)90027-4. [DOI] [PubMed] [Google Scholar]
  18. Johnson M. S., Overington J. P., Blundell T. L. Alignment and searching for common protein folds using a data bank of structural templates. J Mol Biol. 1993 Jun 5;231(3):735–752. doi: 10.1006/jmbi.1993.1323. [DOI] [PubMed] [Google Scholar]
  19. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  20. Karlin A., Akabas M. H. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron. 1995 Dec;15(6):1231–1244. doi: 10.1016/0896-6273(95)90004-7. [DOI] [PubMed] [Google Scholar]
  21. Karlin S., Zuker M., Brocchieri L. Measuring residue associations in protein structures. Possible implications for protein folding. J Mol Biol. 1994 Jun 3;239(2):227–248. doi: 10.1006/jmbi.1994.1365. [DOI] [PubMed] [Google Scholar]
  22. Kohda D., Hatanaka H., Odaka M., Mandiyan V., Ullrich A., Schlessinger J., Inagaki F. Solution structure of the SH3 domain of phospholipase C-gamma. Cell. 1993 Mar 26;72(6):953–960. doi: 10.1016/0092-8674(93)90583-c. [DOI] [PubMed] [Google Scholar]
  23. Kohda D., Terasawa H., Ichikawa S., Ogura K., Hatanaka H., Mandiyan V., Ullrich A., Schlessinger J., Inagaki F. Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2. Structure. 1994 Nov 15;2(11):1029–1040. doi: 10.1016/s0969-2126(94)00106-5. [DOI] [PubMed] [Google Scholar]
  24. Kuhse J., Laube B., Magalei D., Betz H. Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry. Neuron. 1993 Dec;11(6):1049–1056. doi: 10.1016/0896-6273(93)90218-g. [DOI] [PubMed] [Google Scholar]
  25. Langosch D., Laube B., Rundström N., Schmieden V., Bormann J., Betz H. Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia. EMBO J. 1994 Sep 15;13(18):4223–4228. doi: 10.1002/j.1460-2075.1994.tb06742.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lee J. O., Rieu P., Arnaout M. A., Liddington R. Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 1995 Feb 24;80(4):631–638. doi: 10.1016/0092-8674(95)90517-0. [DOI] [PubMed] [Google Scholar]
  27. Lüthy R., Bowie J. U., Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature. 1992 Mar 5;356(6364):83–85. doi: 10.1038/356083a0. [DOI] [PubMed] [Google Scholar]
  28. Machold J., Utkin Y., Kirsch D., Kaufmann R., Tsetlin V., Hucho F. Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7282–7286. doi: 10.1073/pnas.92.16.7282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nishikawa K., Matsuo Y. Development of pseudoenergy potentials for assessing protein 3-D-1-D compatibility and detecting weak homologies. Protein Eng. 1993 Nov;6(8):811–820. doi: 10.1093/protein/6.8.811. [DOI] [PubMed] [Google Scholar]
  30. Noble M. E., Musacchio A., Saraste M., Courtneidge S. A., Wierenga R. K. Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin. EMBO J. 1993 Jul;12(7):2617–2624. doi: 10.2210/pdb1shf/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rajendra S., Lynch J. W., Pierce K. D., French C. R., Barry P. H., Schofield P. R. Mutation of an arginine residue in the human glycine receptor transforms beta-alanine and taurine from agonists into competitive antagonists. Neuron. 1995 Jan;14(1):169–175. doi: 10.1016/0896-6273(95)90251-1. [DOI] [PubMed] [Google Scholar]
  32. Rajendra S., Lynch J. W., Pierce K. D., French C. R., Barry P. H., Schofield P. R. Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor. J Biol Chem. 1994 Jul 22;269(29):18739–18742. [PubMed] [Google Scholar]
  33. Rajendra S., Vandenberg R. J., Pierce K. D., Cunningham A. M., French P. W., Barry P. H., Schofield P. R. The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element. EMBO J. 1995 Jul 3;14(13):2987–2998. doi: 10.1002/j.1460-2075.1995.tb07301.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rickert K. W., Imperiali B. Analysis of the conserved glycosylation site in the nicotinic acetylcholine receptor: potential roles in complex assembly. Chem Biol. 1995 Nov;2(11):751–759. doi: 10.1016/1074-5521(95)90103-5. [DOI] [PubMed] [Google Scholar]
  35. Russell R. B., Breed J., Barton G. J. Conservation analysis and structure prediction of the SH2 family of phosphotyrosine binding domains. FEBS Lett. 1992 Jun 8;304(1):15–20. doi: 10.1016/0014-5793(92)80579-6. [DOI] [PubMed] [Google Scholar]
  36. Ryan S. G., Buckwalter M. S., Lynch J. W., Handford C. A., Segura L., Shiang R., Wasmuth J. J., Camper S. A., Schofield P., O'Connell P. A missense mutation in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor in the spasmodic mouse. Nat Genet. 1994 Jun;7(2):131–135. doi: 10.1038/ng0694-131. [DOI] [PubMed] [Google Scholar]
  37. Sander C., Schneider R. Database of homology-derived protein structures and the structural meaning of sequence alignment. Proteins. 1991;9(1):56–68. doi: 10.1002/prot.340090107. [DOI] [PubMed] [Google Scholar]
  38. Schmieden V., Kuhse J., Betz H. Agonist pharmacology of neonatal and adult glycine receptor alpha subunits: identification of amino acid residues involved in taurine activation. EMBO J. 1992 Jun;11(6):2025–2032. doi: 10.1002/j.1460-2075.1992.tb05259.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schmieden V., Kuhse J., Betz H. Mutation of glycine receptor subunit creates beta-alanine receptor responsive to GABA. Science. 1993 Oct 8;262(5131):256–258. doi: 10.1126/science.8211147. [DOI] [PubMed] [Google Scholar]
  40. Shenkin P. S., Yarmush D. L., Fine R. M., Wang H. J., Levinthal C. Predicting antibody hypervariable loop conformation. I. Ensembles of random conformations for ringlike structures. Biopolymers. 1987 Dec;26(12):2053–2085. doi: 10.1002/bip.360261207. [DOI] [PubMed] [Google Scholar]
  41. Shiang R., Ryan S. G., Zhu Y. Z., Hahn A. F., O'Connell P., Wasmuth J. J. Mutations in the alpha 1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nat Genet. 1993 Dec;5(4):351–358. doi: 10.1038/ng1293-351. [DOI] [PubMed] [Google Scholar]
  42. Taylor W. R. Hierarchical method to align large numbers of biological sequences. Methods Enzymol. 1990;183:456–474. doi: 10.1016/0076-6879(90)83031-4. [DOI] [PubMed] [Google Scholar]
  43. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thornton J. M., Gardner S. P. Protein motifs and data-base searching. Trends Biochem Sci. 1989 Jul;14(7):300–304. doi: 10.1016/0968-0004(89)90069-8. [DOI] [PubMed] [Google Scholar]
  45. Unwin N. Acetylcholine receptor channel imaged in the open state. Nature. 1995 Jan 5;373(6509):37–43. doi: 10.1038/373037a0. [DOI] [PubMed] [Google Scholar]
  46. Unwin N. Nicotinic acetylcholine receptor at 9 A resolution. J Mol Biol. 1993 Feb 20;229(4):1101–1124. doi: 10.1006/jmbi.1993.1107. [DOI] [PubMed] [Google Scholar]
  47. Unwin N. Projection structure of the nicotinic acetylcholine receptor: distinct conformations of the alpha subunits. J Mol Biol. 1996 Apr 5;257(3):586–596. doi: 10.1006/jmbi.1996.0187. [DOI] [PubMed] [Google Scholar]
  48. Vandenberg R. J., Rajendra S., French C. R., Barry P. H., Schofield P. R. The extracellular disulfide loop motif of the inhibitory glycine receptor does not form the agonist binding site. Mol Pharmacol. 1993 Jul;44(1):198–203. [PubMed] [Google Scholar]
  49. Vogt G., Etzold T., Argos P. An assessment of amino acid exchange matrices in aligning protein sequences: the twilight zone revisited. J Mol Biol. 1995 Jun 16;249(4):816–831. doi: 10.1006/jmbi.1995.0340. [DOI] [PubMed] [Google Scholar]
  50. Wilson K. P., Shewchuk L. M., Brennan R. G., Otsuka A. J., Matthews B. W. Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9257–9261. doi: 10.1073/pnas.89.19.9257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Worley K. C., Wiese B. A., Smith R. F. BEAUTY: an enhanced BLAST-based search tool that integrates multiple biological information resources into sequence similarity search results. Genome Res. 1995 Sep;5(2):173–184. doi: 10.1101/gr.5.2.173. [DOI] [PubMed] [Google Scholar]
  52. Xu X., Matsuno-Yagi A., Yagi T. DNA sequencing of the seven remaining structural genes of the gene cluster encoding the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans. Biochemistry. 1993 Jan 26;32(3):968–981. doi: 10.1021/bi00054a030. [DOI] [PubMed] [Google Scholar]

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