Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

Biochem J. 2013 Sep 1;454(2):311-21. doi: 10.1042/BJ20130638.

Abstract

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr¹⁸⁴ in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr¹⁸⁴ depends on local residues, we generated mutations in an α7/5HT(3A) (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured ¹²⁵I-labelled α-btx binding. The results show that mutations of individual residues near Tyr¹⁸⁴ do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr¹⁸⁴ to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr¹⁸⁴ to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr¹⁸⁴ and local residues contributes to high-affinity subtype-selective α-btx binding.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • Bungarotoxins / chemistry
  • Bungarotoxins / metabolism*
  • Bungarus
  • HEK293 Cells
  • Humans
  • Ligands
  • Models, Molecular*
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Neurotoxins / chemistry
  • Neurotoxins / metabolism*
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Receptors, Nicotinic / chemistry
  • Receptors, Nicotinic / genetics
  • Receptors, Nicotinic / metabolism*
  • Receptors, Serotonin, 5-HT3 / chemistry
  • Receptors, Serotonin, 5-HT3 / genetics
  • Receptors, Serotonin, 5-HT3 / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Reptilian Proteins / chemistry
  • Reptilian Proteins / metabolism*
  • Tyrosine / chemistry*
  • alpha7 Nicotinic Acetylcholine Receptor

Substances

  • Bungarotoxins
  • Chrna7 protein, human
  • HTR3A protein, human
  • Ligands
  • Mutant Proteins
  • Neurotoxins
  • Peptide Fragments
  • Protein Isoforms
  • Receptors, Nicotinic
  • Receptors, Serotonin, 5-HT3
  • Recombinant Fusion Proteins
  • Reptilian Proteins
  • alpha7 Nicotinic Acetylcholine Receptor
  • Tyrosine