Moshe Balass

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

A Beta-Hairpin Structure in a 13-mer Peptide that Binds Alpha-Bungarotoxin with High Affinity and Neutralizes its Toxicity

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

Design and synthesis of peptides that bind α-bungarotoxin with high affinity

Abstract Background: α-Bungarotoxin (α-BTX) is a highly toxic snake venom α-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind α-BTX with high affinity, and inhibit its interaction with AChR with an IC 50 of 2 nM. The design of these peptides was based on a lead peptide with an IC 50 of 3×10 −7 M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. Results: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of α-BTX with the lead peptide, as well as structure–function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC 50 values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against α-BTX lethality. Conclusions: Synthetic peptides with high affinity to α-BTX may be used as potential lead compounds for developing effective antidotes against α-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.

The Binding Site of Acetylcholine Receptor

Abstract: Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) α‐subunit for the analysis of the binding site of the AChR. A 13‐mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by α‐bungarotoxin (α‐BTX) from a phage‐display peptide library. The solution structure of a complex between this library‐lead peptide and α‐BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure‐function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to α‐BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high‐affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of α‐BTX to the AChR with an IC50 of 2 nM. The solution and crystal structures of complexes of α‐BTX with HAP were solved, demonstrating that the HAP fits snugly to α‐BTX and adopts a β‐hairpin conformation. The X‐ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that α‐BTX wraps around the receptor binding‐site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand‐binding site. Our proposed model explains the strong antagonistic activity of α‐BTX and accommodates much of the biochemical data on the mode of interaction of α‐BTX with the AChR.

Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind alpha-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the alpha-BTX/AChR system yielded 13-mer peptides that bind to alpha-BTX with high affinity and are potent inhibitors of alpha-BTX binding to AChR (IC(50) of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel beta-hairpin structure when bound to alpha-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.

A cyclic peptide with high affinity to α-bungarotoxin protects mice from the lethal effect of the toxin

Employing a combinatorial phage-peptide library, we previously identified the peptide MRYYESSLKSYPD (designated, library-peptide) that binds the snake toxin alpha-bungarotoxin (alpha-BTX) with a moderate binding constant of 10(-6)M (Balass et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6054-6058). Under the experimental conditions employed, we found that the library-peptide did not protect mice from alpha-BTX lethality when injected concomitantly with the toxin. In order to improve the affinity of the peptide to alpha-BTX, we designed and synthesized the peptide CRYYESSLKSYCD (Met1 and Pro12 were replaced by cysteines), which following oxidation creates a single disulfide bond and forms a cyclic structure. The design of the cyclic peptide was based on our previous NMR analysis of the library-peptide/alpha-BTX complex (Scherf et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6059-6064). The cyclic peptide binds alpha-BTX with affinity two orders of magnitude higher than that of the linear library selected peptide. Whereas the library peptide was ineffective, the cyclic peptide conferred protection from alpha-BTX lethality in mice, even when given 1h after the toxin injection. The cyclic peptide conferred complete protection from alpha-BTX lethality in mice when administered 40min prior to toxin injection. However, experiments with the whole venom of the snake Bungarus multicinctus showed that protection could be achieved only when the cyclic peptide was administered concomitantly with the venom.