Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors.
Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the alpha-epsilon than to the alpha-delta binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the alpha-epsilon interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse epsilon subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat epsilon) and 115 (Tyr in mouse, Ser in rat epsilon) converts Wtx-1 affinity for the alpha-epsilon interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both alpha and epsilon subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.
Molles
Brian E
BE
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA. bmolles@ucsd.edu
Rezai
Parastoo
P
Kline
Eric F
EF
McArdle
Joseph J
JJ
Sine
Steven M
SM
Taylor
Palmer
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003435:Crotalid Venoms / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection
D003435:Crotalid Venoms; D018076:DNA, Complementary; D008024:Ligands; D011978:Receptors, Nicotinic; C077895:waglerin