Abstract
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.
Highlights
Of nearly 300 kDa with 20 membrane spans
These small proteins of 57– 80 amino acids are commonly called “3-fingered” snake toxins for their characteristic three loop topology, with each of the three fingers extending from a core “knuckle” region consisting of four conserved disulfide bonds
The muscle nicotinic receptor [1,2,3] contains five polypeptide subunits arranged with radial symmetry around a central pore
Summary
Synthesis and Purification of Waglerin-1—The crude peptides, synthesized by the American Peptide Company (San Jose, CA) or Synpep (Dublin, CA) were dissolved to 0.8 mg/ml in 30 mM Tris-HCl, pH 8.2– 8.5, sterile-filtered and left overnight at room temperature to form the single intramolecular disulfide. A 2-ml injection of the peptide solution was loaded onto a 5-ml high performance liquid chromatography sample loop and purified on a 10- ϫ 250-mm semipreparative C18 column (Vydac) and eluted using a 0.1% trifluoroacetic acid/70% acetonitrile solvent system changing at 1% for Solvent B every 3 min. Complementary synthetic oligonucleotides (Sigma/Genosys, The Woodlands, TX) containing the desired mutation were ligated into the cDNA at unique restriction sites flanking the mutated region. If convenient restriction sites were not available, the Stratagene double primer method was used In this procedure, complementary oligonucleotides containing the desired mutation served as primers in a PCR amplification reaction containing the wild-type plasmid and Pfu polymerase. Waglerin dissociation constants were determined from the fractional reduction of the initial rate of 125I-␣-BgTx binding. Nonspecific binding was determined by incubating the cells with 10 mM carbachol before reacting with 125I-␣-BgTx
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