Orientation of alpha-neurotoxin at the subunit interfaces of the nicotinic acetylcholine receptor.
The alpha-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. Because the alpha-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.
Malany
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Department of Pharmacology, 0636, University of California at San Diego, La Jolla, California 92093, USA.
Osaka
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Sine
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Taylor
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002460:Cell Line; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism
D003039:Cobra Neurotoxin Proteins; C112113:NmmI protein, Naja mossambica mossambica; D011978:Receptors, Nicotinic