Acetylcholine receptor-alpha-bungarotoxin interactions: determination of the region-to-region contacts by peptide-peptide interactions and molecular modeling of the receptor cavity.

Abstract

In previous studies from this laboratory, the binding regions of a-neurotoxins on human and Torpedo acetylcholine (AcCho) receptors (AcChoRs) and the binding regions for the receptor on the toxin were characterized with synthetic peptides of the respective molecules. In the present work, peptides representing the active regions of one molecule are each allowed to bind to each of the active-region peptides of the other molecule. Thus, the interaction of three abungarotoxin (a-BTX) synthetic loop peptides with four synthetic peptides representing the toxin-binding regions on human AcChoR permitted the determination ofthe region-region interactions between a-BTX and the human receptor. Based on the known three-dimensional structure of the toxin, the active peptides of the receptor were then assembled to their appropriate toxin-contact regions by computer model building and energy minimization. This allowed the three-dimensional con- (10) receptors. Conversely, the binding sites for AcChoR on a-bungarotoxin (a-BTX) were mapped by synthetic peptides representing each of the a-BTX loops (11, 12). The present paper describes an approach for studying the details of protein-protein recognition. Each of the active peptides of one protein is allowed to interact with each of the active peptides of the other protein. Based on relative binding affinities of peptide-peptide interactions, the position of AcChoR peptides relative to the a-BTX molecule was assessed. The receptor peptides were docked onto the appropriate regions of a-BTX, whose three-dimensional (3-D) structure is known (13), by computer graphics and energy minimization, thus allowing a 3-D model to be constructed of the binding-site cavity for the toxin on human AcChoR. MATERIALS AND METHODSMaterials. Synthesis, purification, and characterization of the human AcChoR a-chain peptides have been reported (10). The a-neurotoxin binding regions on human AcChoR reside (10) in the peptides shown in Fig. 1. These peptides were used in the present work. The receptor-binding regions on a-BTX are present in the three loop peptides shown in Fig. 1 (12). The synthesis, purification, and characterization ofthe monomeric forms of the three cyclic peptides (Fig. 1) have been described (12). a-BTX and its synthetic peptides were labeled with iodine-125 by using the chloramine-T method (14). Radioiodinated materials were used immediately after labeling. The specific activities of the labeled peptides were: LIN, 3.1 x 103 cpm/pmol; L2, 2.4 x 103 cpm/pmol; and L3E, 2.1 x 103 cpm/pmol.Binding Studies. tTo whom reprint requests should be addressed at:

Localization and synthesis of the hormone-binding regions of the human thyrotropin receptor.

“…For example, the binding of a-neurotoxins to acetylcholine receptor involves several contact regions on the receptor (21,24,25) and on the neurotoxin (26). It has recently been shown (27) that the binding regions on acetylcholine receptor form a single cavity within which the toxin fits. In addition to the main binding regions within hTSHR peptides 12-30 and 324-344, the lower activities in peptides Table 2.…”

Epitope-specific suppression of antibody response in experimental autoimmune myasthenia gravis by a monomethoxypolyethylene glycol conjugate of a myasthenogenic synthetic peptide.

“…The sequence of AcChoRa region between residues 125 and 148 is highly conserved among species. It binds AcCho (3) and contains a universal binding region for long and short a neurotoxins (12,18,22,23). Because of its direct involvement in the binding of AcCho, and since the affinity of the antibodies to the receptor is several orders of magnitude greater than that of AcCho, the antibodies are capable of effectively blocking the AcCho binding site.…”

Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I

“…Each NT-1 mAb recognized two, non-contiguous peptides of NT-1 located within the central loop, which is reportedly important in the binding of snake venom postsynaptic neurotoxins to AchR (Dufton and Hider, 1983;Love and Stroud, 1986;McDaniel et al, 1987;Ruan et al, 1990). To examine further the cross-reactivity of NT-1 mAbs with a-BT, and the non-reactivity of a-CT, computer models of the three toxins were generated using X-ray crystallography co-ordinates of a-BT and a-CT (Love and Stroud, 1986;Betzel et al, 1991).…”

“…Neutralizing epitopes, Ser34-Lys38 (Kase et al, 1989) and Ser34-Glu41 (Chuang et al, 1989), identified by different mAbs against a-BT are located within the central loop which intimately contacts the asubunit of AchR with high affinity (Low, 1979;Love and Stroud, 1986;McDaniel et al, 1987;Ruan et al, 1990). Only one study describes any cross-reactivity, but not neutralization, of other long-chain neurotoxins with anti-(a-BT) mAbs (Kase et al, 1989).…”