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The -Bungarotoxin Binding Site on the Nicotinic Acetylcholine

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The -Bungarotoxin Binding Site on the Nicotinic Acetylcholine Proc. Natl. Acad. Sci. USA Vol. 94, pp. 6054–6058, June 1997 Biochemistry The a-bungarotoxin binding site on the nicotinic acetylcholine receptor: Analysis using a phage–epitope library MOSHE BALASS*†,EPHRAIM KATCHALSKI-KATZIR*, AND SARA FUCHS†‡ Departments of *Membrane Research and Biophysics and †Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel Contributed by Ephraim Katchalski-Katzir, April 9, 1997 ABSTRACT The nicotinic acetylcholine receptor (Ac- short synthetic peptides containing cysteines 192 and 193 bind ChoR) is a ligand-gated ion channel that is activated upon a-BTX specifically, though with moderate affinity (12, 13). binding of acetylcholine. a-Neurotoxins, in particular a-bun- Major components of this binding were shown to be contained garotoxin (a-BTX), bind specifically and with high affinity to within a synthetic dodecapeptide corresponding to residues the AcChoR and compete with binding of the natural ligand. 185–196 of the Torpedo AcChoR a-subunit (13, 14). NMR We employed a 15-mer phage-display peptide library to select analysis indicated that five residues in this dodecapeptide epitopes reacting with a-BTX. Phages bearing the motif (HWVYY, residues 186–190) are in contact with a-BTX (15). YYXSSL as a consensus sequence were found to bind with high Additional evidence for localization of the binding site for affinity to a-BTX. The library-derived peptide (MRYYES- a-BTX within this particular region of the a-subunit came SLKSYPD) bears amino acid sequence similarities to a region from recent analysis of the presumed ligand-binding site of of the a-subunit of the Torpedo muscle AcChoR, as well as of AcChoRs from animals that are resistant to a-BTX (10, other muscle and neuronal AcChoRs that bind -BTX. The a 16–18). This analysis indicated unique sequence differences library-derived peptide and the corresponding peptides con- between toxin-resistant and toxin-sensitive receptors, concen- taining residues 187–199 of the Torpedo AcChoR a-subunit a (WVYYTCCPDTPYL), as well as peptides analogous to the trated within a very small region of the -subunit, close to cysteines 192 and 193. above region in the neuronal AcChoR (e.g., human a7; ER- FYECCKEPYPD) that binds a-BTX, inhibit the binding of Another approach in searching for a peptide that interacts 26 specifically with a-BTX is to employ random peptide libraries a-BTX to the intact Torpedo AcChoR with IC50 values of 10 M. A synthetic peptide from a neuronal AcChoR that does not (19, 20). One advantage of such an approach is that the selector molecule is not ‘‘committed’’ to any target molecule known to bind a-BTX (e.g., human a2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide interact with it and is allowed to select peptides from a from the a-BTX-binding protein (a7)—i.e., Lys in a2 and Tyr completely random collection of peptide-presenting phages. in a7—does not inhibit the binding of a-BTX to Torpedo We have previously employed phage–epitope libraries to study AcChoR. These results indicate the requirement for two the AcChoR and have identified peptide epitopes for two adjacent aromatic amino acid residues for binding to a-BTX. anti-AcChoR monoclonal antibodies (21, 22). In the present study we utilized a 15-mer phage–epitope The nicotinic acetylcholine receptor (AcChoR) is a ligand- library to select a peptide that interacts specifically with gated ion channel that is activated by binding of acetylcholine a-BTX and blocks its interaction with the AcChoR. The (AcCho). In muscle, the functional AcChoR molecule is a library-derived peptide bears a similarity to the ligand-binding pentameric complex of a2bgd subunits, or a2b«d subunits. The region in the a-subunit of both muscle and neuronal a-BTX- ligand-binding site of the AcChoR has been studied extensively binding AcChoRs. Identification of this peptide confirms the and found to be located on the a-subunit, in the vicinity of localization of the binding site within the receptor molecule cysteines 192 and 193. It was further shown that the ligand- and allows a more precise determination of the boundaries of binding domains are formed at the interfaces between the a-g the binding site. This peptide could also be employed as a lead and a-d subunits (for reviews, see 1–3). Affinity-labeling compound in the search for effective antidotes against toxin experiments indicated that within the a-subunit, cysteines 192 intoxication. In this connection, the constrained conformation and 193, which form an intrachain disulfide in the intact attained by the peptide in a complex with a-BTX as revealed receptor (4), and the conserved aromatic residues, Tyr-93, by NMR studies (see the companion article, ref. 23), should Trp-149, Tyr-190, and Tyr-198, are labeled by derivatives of obviously be taken into consideration. agonists or antagonists (4–9) and are thus within the binding site, or very close to it. The curarimetic snake a-neurotoxins, such as a-bungaro- MATERIALS AND METHODS toxin (a-BTX), are potent competitive inhibitors of AcChoR Epitope Library. The 15-mer phage–peptide library em- function and are highly toxic due to functional blockade of ployed in this study was provided by James J. Devlin (Cetus) AcChoRs at the neuromuscular junction. Because of the high and was constructed by use of phage M13 as described (20). affinity and specificity of a-BTX for the AcChoR, many studies The library consists of about 2 3 107 different 15-residue of the AcChoR ligand-binding site have focused on the binding peptide-presenting phages. site for a-BTX. Though the binding sites for AcCho and a-BTX probably overlap, structural requirements for their Biotinylation of a-Bungarotoxin. HPLC-purified a-BTX binding may differ, and amino acid residues critical for binding from the snake venom of Bungarus multicinctus was purchased of the toxin may not be the same as those critical for binding from Sigma. For biotinylation, 100 mgofa-BTX in 100 mlof of AcCho (10, 11). Previous studies have demonstrated that 0.1 M NaHCO3 (pH 8.6) were incubated for2hatroom The publication costs of this article were defrayed in part by page charge Abbreviations: AcCho, acetylcholine; AcChoR, AcCho receptor; a-BTX, a-bungarotoxin; bio-a-BTX, biotinylated a-BTX; HMB, half payment. This article must therefore be hereby marked ‘‘advertisement’’ in maximal binding; TyP, Tween 20 in PBS. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed. e-mail: © 1997 by The National Academy of Sciences 0027-8424y97y946054-5$2.00y0 [email protected]. 6054 Downloaded by guest on September 27, 2021 Biochemistry: Balass et al. Proc. Natl. Acad. Sci. USA 94 (1997) 6055 temperature with 5 mg of biotin amidocaproate N-hydroxy- Table 1. Origin and sequence of synthetic peptides studied succinimide ester (Sigma) from a stock solution of 1 mgyml in Peptide Sequence dimethylformamide, and then dialyzed at 4°C against PBS (0.14 M NaCly0.01 M phosphate buffer, pH 7.4). Library-derived peptides Isolation of Peptide-Presenting Phages from the Phage– 15-mer YMRYYESSLKSYPDW Peptide Library. A sample of the amplified library, containing 17-mer KKYMRYYESSLKSYPDW MRYYESSKLSYPDW 1011 infectious phage particles, was subjected to three rounds 14-mer MRYYESSLKSYPD of selection (panning) and amplification. In the first round, 13-mer* 12-mer MRYYESSLKSYP 1011 phage particles were incubated overnight at 4°C with 20 11-mer MRYYESSLKSY ng ml biotinylated-a-BTX (bio-a-BTX) in 40 ml of 0.5% BSA y Peptides derived from sequences of Torpedo AcChoR in PBS. The phages were diluted with 1 ml 0.5% Tween 20 in a1 187-199 WVYYTCCPDTPYL PBS (TyP) and allowed to react for 30 min at room temper- a1 185-196 KHWVYYTCCPDT ature with streptavidin (10 mgyml)-coated Petri dishes (60 Peptides derived from sequences of neuronal mm) with gentle shaking. Unbound phages were removed by a-BTX-binding AcChoRs extensive washing (10 times, 10 min each) in TyP and the Rat a7 EKFYECCKEPYPD remaining phages were eluted with 0.8 ml 0.1 M HCl, titrated Chick a8 ELYYECCKEPYPD to pH 2.2 with glycine. The eluate was neutralized with 48 ml Human a7 ERFYECCKEPYPD of 2 M Tris base and used to infect Escherichia coli K91 cells. Peptide derived from the sequence of a neuronal The phage eluted in the first round were amplified and AcChoR that does not bind a-BTX subjected to a second round of affinity purification with the Human a2 ERKYECCKEPYPD bio-a-BTX at 2 ngyml. Phage from the second round were *Library-derived peptide. amplified and subjected to a third round with 0.2 ngyml bio-a-BTX. The progressively decreasing a-BTX concentra- RESULTS tions used in the second and third rounds, and the use of excess phages, were intended to facilitate selection for high-affinity Selection of Peptide-Presenting Phages by Bio-a-BTX. To a epitopes. After three pannings, individual bacterial colonies isolate peptides representing the -BTX binding site within AcChoRs or other proteins that may interact specifically with containing amplified phage clones were grown in a microtiter a-BTX, we screened a 15-mer phage–peptide library with plate and screened by ELISA for their ability to specifically bio-a-BTX. After three cycles of panning and phage amplifi- bind bio-a-BTX, essentially as described (21). Phage DNAs cation, 90 individually isolated bacterial colonies were grown were sequenced to determine the amino acid sequences of in microtiter plates and their phages were assayed for toxin their inserted peptides. binding. ELISA analysis revealed that 60 of these clones a-BTX Binding to Isolated Phages.
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