Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite (original) (raw)
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The binding sire of the acetylcholine receptor from animals resistant to α-neurotoxins
Toxicon, 1995
The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the a-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. In an attempt to elucidate the molecular basis for ligand binding, we investigated the binding site structure of AChR from several animal species which show various degrees of resistance to ~-bungarotoxin (~t-BTX). Fragments of the AChR rt-subunit encoding residues 122-205 from snakes, mongoose, hedgehog, human, shrew, cat and mouse were amplified by reverse transcription and PCR, and then sequenced and expressed in E. coll. All fragments are highly homologous and contain the four cysteines at residues 128, 142, 192 and 193. The snake, mongoose and hedgehog fragments did not bind a-BTX and they all have substitutions at the conserved positions 187 and 189. This indicates that changes at these positions from the aromatic residues Trp and Phe, respectively, to non-aromatic ones, may be sufficient for conferring toxin resistance. The snake and the mongoose AChR have additional substitutions at positions 194 (snake and mongoose) and 197 (mongoose). It should be noted that the human AChR fragment, which is a partial binder and binds ,,-BTX to a lesser extent than the mouse, cat and shrew fragments, also has substitutions at positions 187 and 189 from aromatic to non aromatic residues. To identify further the amino acids participating in a-B'IX binding, a series of point mutations was performed, changing residues at positions 187, 189, 194, and 197 each alone or in combination, from mongoose to the mouse sequence. Expression and binding analysis of the mutated fragments provide additional evidence that aromatic amino acid residues at positions 187 and 189, and prolines at positions 194 and 197 are important in creating the appropriate structure allowing e,-BTX binding.
Journal of Biological Chemistry, 1999
We have investigated the molecular determinants responsible for ␣-bungarotoxin (␣Bgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous ␣ subunits, one highly sensitive to ␣Bgtx block (␣1) and the other, ␣Bgtx-insensitive (␣3). By replacing rat ␣3 residues 184-191 with the corresponding region from the Torpedo ␣1 subunit, we introduced a cluster of five ␣1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the ␣3 subunit. Functional activity and ␣Bgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric ␣3 subunit (␣3/␣1[5]) with either rat 2 or 4 subunits. Agonist-evoked responses of ␣3/␣1[5]-containing receptors were blocked by ␣Bgtx with nanomolar affinity (IC 50 values: 41 nM for ␣3/␣1[5]2 and 19 nM for ␣3/␣1[5]4). Furthermore, receptors containing the single point mutation ␣3K189Y acquire significant sensitivity to ␣Bgtx block (IC 50 values: 186 nM for ␣3K189Y2 and 179 nM for ␣3K189Y4). Another ␣3 chimeric subunit, ␣3/␣7[6], similar to ␣3/␣1[5] but incorporating the corresponding residues from the ␣Bgtx-sensitive ␣7 subunit, also conferred potent ␣Bgtx sensitivity to chimeric receptors when co-expressed with the 4 subunit (IC 50 value ؍ 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the ␣Bgtx-sensitive subunits ␣1 and ␣7 play a critical functional role in the interaction of ␣Bgtx with nicotinic acetylcholine receptors sensitive to this toxin.
Neuroscience Letters, 1987
Key wordsv 7-Bungarotoxin; Acetylcholine receptor; Cholinergic binding site; Cholinergic ligand: Naja naja siarnensis ~-toxin; PI 5 toxin In order to study where the binding site of cholinergic agents is in the sequence of the ~-subunit of nicotinic acetylcholine receptor (AChR), we have synthetized 3 peptides with an aminoacid sequence corresponding to the following sequences of the ~-subunit of Torpedo ealifornica AChR: 125 143, 158 167, [Lys] 188 201. For binding studies the peptides were immobilized on Sepharose 4B. Only the peptide [Lys] 188 201 binds t251-cc-bungarotoxin (c~-Bgtx) with Kd of 1.03 ,uM. The binding of 1251-c~-Bgtx to the peptide is reduced by 85% after reduction of the S S bridge present between 192 193 cysteines indicating that an intact disulfide bond is important for toxin binding. The ~251-~-Bgtx binding is inhibited by curare, decamethonium, hexamethonium but not by carbamylcholine and Naja naja siamensis ~-toxin and P15 toxin. All these data provide direct evidence that the sequence 188 201 of the ~-subunit of AChR binds ~-Bgtx and that this binding has a pharmacological profile similar to that of nicotinic acetylcholine receptor.
Biochemistry, 1994
In the a subunit of the Torpedo nicotinic cholinergic receptor (AChR), a sequence region surrounding a pair of adjacent cysteinyl residues at positions 192 and 193 contributes to a binding site for cholinergic ligands, including the snake a-neurotoxins. Synthetic and biosynthetic peptides corresponding to this region bind a-bungarotoxin (a-BTX) in the absence of other structural components of the AChR and, therefore, represent a "prototope" for a-BTX. Using synthetic peptides corresponding to the complete AChR a subunits of Torpedo electroplax and mammalian muscle, we previously defined a sequence segment corresponding to a universal prototope for a-BTX binding between amino acid residues 18 1 and 200 [Conti
Biochemical Journal, 2003
The neuronal α7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin α-bungarotoxin (α-Bgt). Fine mapping of the α-Bgt-binding site on the human α7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human α7 subunit (residues 1–206). Screening of these peptides for 125I-α-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186–197, which exhibited the best 125I-α-Bgt binding, and one at residues 159–165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the α7(186–197) peptide was almost completely inhibited by unlabelled α-Bgt or d-tubocurarine. Alanine substitutions within the sequence 186–198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the α-Bgt binding site of the α1 subunit (residues 188–200 in Torpedo AChR). In competiti...