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. 1990 May;87(9):3378–3382. doi: 10.1073/pnas.87.9.3378

Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives.

B Chatrenet 1, O Trémeau 1, F Bontems 1, M P Goeldner 1, C G Hirth 1, A Ménez 1
PMCID: PMC53903  PMID: 2333287

Abstract

We have defined the molecular environment of a snake neurotoxin interacting with the high- and low-affinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys-47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the high-affinity site, the probes on Lys-15 and Lys-47 predominantly reacted with the high-affinity site of the AcChoR alpha subunit whereas the probe on Lys-51 reacted with the delta subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the alpha chain and the beta chain whereas those on Lys-15 and Lys-51 reacted with the gamma and delta chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amit A. G., Mariuzza R. A., Phillips S. E., Poljak R. J. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science. 1986 Aug 15;233(4765):747–753. doi: 10.1126/science.2426778. [DOI] [PubMed] [Google Scholar]
  2. Boulain J. C., Ménez A., Couderc J., Faure G., Liacopoulos P., Fromageot P. Neutralizing monoclonal antibody specific for Naja nigricollis toxin alpha: preparation, characterization, and localization of the antigenic binding site. Biochemistry. 1982 Jun 8;21(12):2910–2915. doi: 10.1021/bi00541a016. [DOI] [PubMed] [Google Scholar]
  3. Boulain J. C., Ménez A. Neurotoxin-specific immunoglobulins accelerate dissociation of the neurotoxin-acetylcholine receptor complex. Science. 1982 Aug 20;217(4561):732–733. doi: 10.1126/science.7100919. [DOI] [PubMed] [Google Scholar]
  4. Brisson A., Unwin P. N. Quaternary structure of the acetylcholine receptor. Nature. 1985 Jun 6;315(6019):474–477. doi: 10.1038/315474a0. [DOI] [PubMed] [Google Scholar]
  5. Dennis M., Giraudat J., Kotzyba-Hibert F., Goeldner M., Hirth C., Chang J. Y., Lazure C., Chrétien M., Changeux J. P. Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site. Biochemistry. 1988 Apr 5;27(7):2346–2357. doi: 10.1021/bi00407a016. [DOI] [PubMed] [Google Scholar]
  6. Dufton M. J., Hider R. C. Conformational properties of the neurotoxins and cytotoxins isolated from Elapid snake venoms. CRC Crit Rev Biochem. 1983;14(2):113–171. doi: 10.3109/10409238309102792. [DOI] [PubMed] [Google Scholar]
  7. Endo T., Tamiya N. Current view on the structure-function relationship of postsynaptic neurotoxins from snake venoms. Pharmacol Ther. 1987;34(3):403–451. doi: 10.1016/0163-7258(87)90002-7. [DOI] [PubMed] [Google Scholar]
  8. Faure G., Boulain J. C., Bouet F., Montenay-Garestier T., Fromageot P., Ménez A. Role of indole and amino groups in the structure and function of Naja nigricollis toxin alpha. Biochemistry. 1983 Apr 26;22(9):2068–2076. doi: 10.1021/bi00278a006. [DOI] [PubMed] [Google Scholar]
  9. Fryklund L., Eaker D. The complete covalent structure of a cardiotoxin from the venom of Naja nigricollis (African black-necked spitting cobra). Biochemistry. 1975 Jul;14(13):2865–2871. doi: 10.1021/bi00684a012. [DOI] [PubMed] [Google Scholar]
  10. Garcia-Borron J. C., Bieber A. L., Martinez-Carrion M. Reductive methylation as a tool for the identification of the amino groups in alpha-bungarotoxin interacting with nicotinic acetylcholine receptor. Biochemistry. 1987 Jul 14;26(14):4295–4303. doi: 10.1021/bi00388a017. [DOI] [PubMed] [Google Scholar]
  11. Hamilton S. L., Pratt D. R., Eaton D. C. Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by alpha-neurotoxin cross-linking. Biochemistry. 1985 Apr 23;24(9):2210–2219. doi: 10.1021/bi00330a015. [DOI] [PubMed] [Google Scholar]
  12. Hucho F. Photoaffinity derivatives of alpha-bungarotoxin and alpha-Naja naja siamensis toxin. FEBS Lett. 1979 Jul 1;103(1):27–32. doi: 10.1016/0014-5793(79)81243-0. [DOI] [PubMed] [Google Scholar]
  13. Ishikawa Y., Menez A., Hori H., Yoshida H., Tamiya N. Structure of snake toxins and their affinity to the acetylcholine receptor of fish electric organ. Toxicon. 1977;15(6):477–488. doi: 10.1016/0041-0101(77)90098-8. [DOI] [PubMed] [Google Scholar]
  14. Kubalek E., Ralston S., Lindstrom J., Unwin N. Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata. J Cell Biol. 1987 Jul;105(1):9–18. doi: 10.1083/jcb.105.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lobel P., Kao P. N., Birken S., Karlin A. Binding of a curarimimetic toxin from cobra venom to the nicotinic acetylcholine receptor. Interactions of six biotinyltoxin derivatives with receptor and avidin. J Biol Chem. 1985 Sep 5;260(19):10605–10612. [PubMed] [Google Scholar]
  17. Low B. W., Preston H. S., Sato A., Rosen L. S., Searl J. E., Rudko A. D., Richardson J. S. Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acetylcholine receptor. Proc Natl Acad Sci U S A. 1976 Sep;73(9):2991–2994. doi: 10.1073/pnas.73.9.2991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ménez A. Molecular immunology of snake toxins. Pharmacol Ther. 1985;30(1):91–113. doi: 10.1016/0163-7258(85)90049-x. [DOI] [PubMed] [Google Scholar]
  19. Nathanson N. M., Hall Z. W. In situ labeling of Torpedo and rat muscle acetylcholine receptor by a photoaffinity derivative of alpha-bungarotoxin. J Biol Chem. 1980 Feb 25;255(4):1698–1703. [PubMed] [Google Scholar]
  20. Neubig R. R., Cohen J. B. Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions. Biochemistry. 1979 Nov 27;18(24):5464–5475. doi: 10.1021/bi00591a032. [DOI] [PubMed] [Google Scholar]
  21. Oswald R. E., Changeux J. P. Crosslinking of alpha-bungarotoxin to the acetylcholine receptor from Torpedo marmorata by ultraviolet light irradiation. FEBS Lett. 1982 Mar 22;139(2):225–229. doi: 10.1016/0014-5793(82)80857-0. [DOI] [PubMed] [Google Scholar]
  22. Popot J. L., Changeux J. P. Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein. Physiol Rev. 1984 Oct;64(4):1162–1239. doi: 10.1152/physrev.1984.64.4.1162. [DOI] [PubMed] [Google Scholar]
  23. Ratnam M., Gullick W., Spiess J., Wan K., Criado M., Lindstrom J. Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding. Biochemistry. 1986 Jul 29;25(15):4268–4275. doi: 10.1021/bi00363a014. [DOI] [PubMed] [Google Scholar]
  24. Rousselet A., Faure G., Boulain J. C., Ménez A. The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody. An electron-spin-resonance study. Eur J Biochem. 1984 Apr 2;140(1):31–37. doi: 10.1111/j.1432-1033.1984.tb08063.x. [DOI] [PubMed] [Google Scholar]
  25. Saitoh T., Oswald R., Wennogle L. P., Changeux J. P. Conditions for the selective labelling of the 66 000 dalton chain of the acetylcholine receptor by the covalent non-competitive blocker 5-azido-[3H]trimethisoquin. FEBS Lett. 1980 Jul 11;116(1):30–36. doi: 10.1016/0014-5793(80)80522-9. [DOI] [PubMed] [Google Scholar]
  26. Sine S. M., Taylor P. Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. J Biol Chem. 1981 Jul 10;256(13):6692–6699. [PubMed] [Google Scholar]
  27. Trémeau O., Boulain J. C., Couderc J., Fromageot P., Ménez A. A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short-chain variants. FEBS Lett. 1986 Nov 24;208(2):236–240. doi: 10.1016/0014-5793(86)81024-9. [DOI] [PubMed] [Google Scholar]
  28. Tzartos S. J., Changeux J. P. Lipid-dependent recovery of alpha-bungarotoxin and monoclonal antibody binding to the purified alpha-subunit from Torpedo marmorata acetylcholine receptor. Enhancement by noncompetitive channel blockers. J Biol Chem. 1984 Sep 25;259(18):11512–11519. [PubMed] [Google Scholar]
  29. Witzemann V., Muchmore D., Raftery M. A. Affinity-directed cross-linking of membrane-bound acetylcholine receptor polypeptides with photolabile alpha-bungarotoxin derivatives. Biochemistry. 1979 Nov 27;18(24):5511–5518. doi: 10.1021/bi00591a039. [DOI] [PubMed] [Google Scholar]
  30. Zeghloul S., Marchot P., Bougis P. E., Ronin C. Selective loss of binding sites for the iodinated alpha-neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes. Eur J Biochem. 1988 Jun 15;174(3):543–550. doi: 10.1111/j.1432-1033.1988.tb14133.x. [DOI] [PubMed] [Google Scholar]

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