Identification of Pairwise Interactions in the α-Neurotoxin-Nicotinic Acetylcholine Receptor Complex through Double Mutant Cycles

Abstract

␣-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the ␣-neurotoxin from Naja mossambica mossambica ( The nicotinic acetylcholine receptor (nAChR) 1 is a member of the large family of neurotransmitter-gated ion channels (for review, see Refs. 1-4). It is composed of five subunits that are arranged in the circular order of ␣␥␣␦␤. The two ligand binding sites reside in the extracellular domain at the ␣␥ and ␣␦ subunit interfaces (5, 6). A high resolution structure is not available for any member of the family of neurotransmitter-gated ion channels. However, within this family the structure of the nAChR has been most extensively characterized through sitedirected labeling (7-12), site-specific mutagenesis (13-23), electron microscopy reconstruction analysis (24, 25), and homology modeling (26). In current models, three discontinuous regions or domains on the ␣-subunit (encompassing residues around 93, 149 -154, and 180 -200) and four regions on the ␥/␦ subunits are thought to participate in the formation of the binding sites (2, 26). Because of sequence differences in the ␦ and ␥ subunits, the binding sites on the receptor are not identical; consequently, many ligands bind to each site with different affinities (15,22,27).The critical tool utilized in the initial identification of the receptor and in subsequent structural analyses is the family of three-fingered snake ␣-neurotoxins (for review, see Refs. 28 and 29), which form high affinity complexes with the receptor; for example, the ␣-bungarotoxin-receptor complex has a K d of ϳ10Ϫ12 . The structure of the ␣-neurotoxins has been solved through nuclear magnetic resonance (30 -32) and x-ray crystallographic studies (33-35). These polypeptides (ϳ7 kDa) are characterized by three large loops which extend from a rigid globular domain held together by 4 or 5 conserved disulfide bonds. Even though ␣-neurotoxin structures have been solved, little is known about the structure of the toxin-receptor complex, and interacting residues have not been identified.In previous work, we delineated residues involved in the binding interaction on both the ␣-neurotoxin, Naja mossambica mossambica (NmmI), and the mouse muscle nAChR interfaces (36). Four residues located on the receptor ␣-subunit and three residues located on the toxin structure were found to contribute significantly to high affinity binding. Even though wild-type NmmI displays an equivalent affinity for the two binding sites on nAChR, we showed through mutational analysis that the energetic contribution of selected residues differed at the two sites. Several of the toxin and receptor mutations studied differentially affected binding at the ␣␦ and ␣␥ sites, resulting in two distinct binding affinities (36). In this study, we have utilized double mutant cycles to explore whether any of these residues are involved in pairwise interact...

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

“…Clearly the loop C that contains highly functional residues conserved in both receptors plays a predominant binding role. The evidence that supports that conclusion includes (i) mutational analyses with short chain (33,34) and long chain (35)(36)(37) toxins, (ii) the use of synthetic receptor peptides of the region 180-200 (38,39), (iii) studies on the resistance of various species to toxins (40,41), (iv) direct affinity labeling experiments (42), and (v) resolution of solution structures of the complexes formed between ␣-Bgtx and peptides (32,(43)(44)(45). Also, the additional binding function observed here for other ␣ and non-␣ loops was postulated for muscular receptors (46)(47)(48).…”

Structure of the interferon-receptor complex determined by distance constraints from double-mutant cycles and flexible docking

“…DMC data have previously been used for rough docking of a peptide inhibitor to a K ϩ channel pore, a peptide to an antibody, and a ligand to a receptor (21)(22)(23). The innovation in the present work is to adapt the mutagenesis information into NMR-style inter-residue distance constraints and use these constraints to drive the docking computationally.…”

Recombinant Expression of α‐Bungarotoxin in Pichia pastoris Facilitates Identification of Mutant Toxins Engineered to Recognize Neuronal Nicotinic Acetylcholine Receptors

“…Intracellular expression of ␣-neurotoxins as fusion proteins in E. coli results in slightly higher yields of purified toxin. Mé-nez and co-workers reported levels of 0.2-2 mg/L for wild-type and mutant Ea forms (Ducancel et al, 1989;Pillet et al, 1993;Tremeau et al, 1995;Drevet et al, 1997), whereas Taylor and co-workers reported 0.5-1.5 mg/L for wild-type and mutant NmmI forms (Ackermann and Taylor, 1997; Ackermann et al, 1998). Both Ea and NmmI are short ␣-neurotoxins, however, lacking the fifth disulfide found in ␣Bgtx.…”

“…Ménez and co-workers have characterized 40 mutations at 36 of the 62 residues in Ea but found that only 10 sites appeared critical for nAChR binding (Pillet et al, 1993;Tremeau et al, 1995). Taylor and co-workers have studied four charged residues of NmmI and, extending their analysis to include mutations of four residues in the nAChR ␣-subunit sequence (a double mutant cycle analysis), have identified contacts between this short ␣-neurotoxin and the native receptor (Ackermann and Taylor, 1997;Ackermann et al, 1998). Some of the residues of Bgtx that give it specificity for certain neuronal nAChRs have also been identified (Fiordalisi et al, 1994;Chiappinelli et al, 1996), and the importance of the conserved disulfide bonds in this toxin has been evaluated by mutation of pairs of cysteines (Grant et al, 1998).…”

“…Erabutoxin a [Ea (Ducancel et al, 1989;Pillet et al, 1993;Tremeau et al, 1995;Drevet et al, 1997)], Naja mossambica mossambica toxin I [NmmI (Ackermann and Taylor, 1997;Ackermann et al, 1998)], Bgtx (Fiordalisi et al, 1994Chiappinelli et al, 1996;Grant et al, 1998), and ␣Bgtx (Rosenthal et al, 1994(Rosenthal et al, , 1999 have been investigated by site-directed mutagenesis. The major goal of many of these studies has been to identify toxin residues that are important in binding to the nAChRs.…”