title: NMR mapping and secondary structure determination of the major acetylcholine receptor alpha-subunit determinant interacting with alpha-bungarotoxin.
abstract: The alpha-subunit of the nicotinic acetylcholine receptor (alphaAChR) contains a binding site for alpha-bungarotoxin (alpha-BTX), a snake-venom-derived alpha-neurotoxin. Previous studies have established that the segment comprising residues 173-204 of alphaAChR 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 alphaAChR determinant interacting with alpha-BTX. Two overlapping synthetic peptides corresponding to segments 179-200 and 182-202 of the alphaAChR were complexed with alpha-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 alpha-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 T(2) measurements. Complete resonance assignment of the 11.2 kDa complex of alpha-BTX bound to the alphaAChR peptide comprising residues 182-202 was obtained at pH 4 using homonuclear 2D NMR spectra measured at 800 MHz. The secondary structures of both alpha-BTX and the bound alphaAChR peptide were determined using 2D (1)H NMR experiments. The peptide folds into a beta-hairpin conformation, in which residues (R)H186-(R)V188 and (R)Y198-(R)D200 form the two beta-strands. Residues (R)Y189-(R)T191 form an intermolecular beta-sheet with residues (B)K38-(B)V40 of the second finger of alpha-BTX. These results accurately pinpoint the alpha-BTX-binding site on the alphaAChR and pave the way to structure determination of this important alphaAChR determinant involved in binding acetylcholine and cholinergic agonists and antagonists.
authors.affiliation: Department of Structural Biology and Chemical Services, The Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: A O
authors.initials: AO
authors.lastname: Samson
authors.forename: J H
authors.initials: JH
authors.lastname: Chill
authors.forename: E
authors.initials: E
authors.lastname: Rodriguez
authors.forename: T
authors.initials: T
authors.lastname: Scherf
authors.forename: J
authors.initials: J
authors.lastname: Anglister
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014101:Torpedo; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D002038:Bungarotoxins; C579166:Chrna7 protein, mouse; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor