Received September 26, 2000

Revised Manuscript Received January 23, 2001

Abstract:

The -subunit of the nicotinic acetylcholine receptor (AChR) contains a binding site for -bungarotoxin (-BTX), a snake-venom-derived -neurotoxin. Previous studies have established that the segment comprising residues 173-204 of AChR contains the major determinant interacting with the toxin, but the precise boundaries of this determinant have not been clearly defined to date. In this study, we applied NMR dynamic filtering to determine the exact sequence constituting the major AChR determinant interacting with -BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the AChR were complexed with -BTX. HOHAHA and ROESY spectra of these complexes acquired with long mixing times highlight the residues of the peptide that do not interact with the toxin and retain considerable mobility upon binding to -BTX. These results, together with changes in the chemical shifts of the peptide protons upon complex formation, suggest that residues 184-200 form the contact region. At pH 4, the molecular mass of the complex determined by dynamic light scattering (DLS) was found to be 11.2 kDa, in excellent agreement with the expected molecular mass of a 1:1 complex, while at pH >5 the DLS measurement of 20 kDa molecular mass indicated dimerization of the complex. These results were supported by T₂ measurements. Complete resonance assignment of the 11.2 kDa complex of -BTX bound to the AChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both -BTX and the bound AChR peptide were determined using 2D ¹H NMR experiments. The peptide folds into a -hairpin conformation, in which residues ^RH186-^RV188 and ^RY198-^RD200 form the two -strands. Residues ^RY189-^RT191 form an intermolecular -sheet with residues ^BK38-^BV40 of the second finger of -BTX. These results accurately pinpoint the -BTX-binding site on the AChR and pave the way to structure determination of this important AChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.

Download the full text: PDF | HTML