Solution Conformation of α-Conotoxin EI, a Neuromuscular Toxin Specific for the α1/δ Subunit Interface of Torpedo Nicotinic Acetylcholine Receptor

Abstract

A high resolution structure of ␣-conotoxin EI has been determined by 1 H NMR spectroscopy and molecular modeling. ␣-Conotoxin EI has the same disulfide framework as ␣4/7 conotoxins targeting neuronal nicotinic acetylcholine receptors but antagonizes the neuromuscular receptor as do the ␣3/5 and ␣A conotoxins. The unique binding preference of ␣-conotoxin EI to the ␣ 1 /␦ subunit interface of Torpedo neuromuscular receptor makes it a valuable structural template for superposition of various ␣-conotoxins possessing distinct receptor subtype specificities. Structural comparison of ␣-conotoxin EI with the ␥-subunit favoring ␣-conotoxin GI suggests that the Torpedo ␦-subunit preference of the former originates from its second loop. Superposition of three-dimensional structures of seven ␣-conotoxins reveals that the estimated size of the toxin-binding pocket in nicotinic acetylcholine receptor is ϳ20 Å (height) ؋ 20 Å (width) ؋ 15 Å (thickness).The nicotinic acetylcholine receptors (nAChRs) 1 are a well studied family of ligand-gated ion channels comprising a diverse set of molecular subtypes (1). The best characterized is the receptor at the neuromuscular junction, with four different subunits in a pentameric array, i.e. (␣ 1 ) 2 ␤ 1␥ ␦. Less well understood and more diverse are the neuronal nAChRs that assemble in exogenous expression systems with a general composition of (␣ m ) 2 (␤ n ) 3 , where m ϭ 2-6 and n ϭ 2-4, or (␣ 7 ) 5 (2, 3). A large variety of ligands bind to such diverse nAChRs via unknown mechanisms. A better understanding of the ligandbinding mechanism would be possible if a suitable three-dimensional structure of nAChR were available. Although cryoelectron microscopic images of nAChR show the spatial arrangement of five subunits of the receptor and some aspects of the ligand-binding pockets (4, 5), they are as yet insufficient for describing ligand-receptor interactions in atomic detail. In this regard, the recently determined crystal structure of acetylcholine-binding protein is expected to provide useful insight into ligand-nAChR interactions (6).Small peptide toxins of Conus origin known as the ␣-and ␣A-conotoxins are highly useful tools for exploring ligandnAChR interactions (7,8). A well defined subgroup of ␣-conotoxins, referred to as the ␣3/5 2 subfamily conotoxins (Table I), are antagonists of neuromuscular receptors and show, except for ␣-conotoxin SI (9, 10), binding preference to the ␣ 1 /␥ subunit interface of Torpedo nAChR. On the other hand, more recently found ␣A-conotoxins differ from the ␣3/5 subfamily conotoxins in the amino acid sequences (11, 12) as well as in their threedimensional structures (13). The ␣A-conotoxins, nevertheless, target neuromuscular receptors. A third subfamily of ␣-conotoxins known as the ␣4/7 subfamily has also been found; members of this subfamily target subtypes of neuronal and muscle nAChRs (14 -17). The ␣4/7 subfamily contains two disulfide bonds like the ␣3/5 subfamily but has a different spacing between the disulfide bonds.Even though a high resolutio...

Analogs of α-Conotoxin MII Are Selective for α6-Containing Nicotinic Acetylcholine Receptors

“…Despite their differences in primary sequence, NMR and X-ray crystallography studies show a high conservation of the structural peptide backbone (Hu et al, 1996(Hu et al, , 1997(Hu et al, , 1998Shon et al, 1997;Hill et al, 1998;Cho et al, 2000;Park et al, 2001;Nicke et al, 2003). It seems that this backbone serves as a scaffold that presents a variety of amino acid side chains leading to differences in specificity.…”

Molecular docking study on the α3β2 neuronal nicotinic acetylcholine receptor complexed with α-Conotoxin GIC

“…Ligand binding modes of these residues have been confirmed in recent homology modeled structures of nAChRs (31) and the x-ray structures of AChBP bound to α-conotoxins (23,25,32) or α-cobratoxin (33). Another strategy for studying nAChR-ligand interactions has been to identify potential receptor-binding pharmacophores within various ligands including α-conotoxins (16,17,19,21,(34)(35)(36)(37). Here, we have generated high-resolution structures of ligand-nAChR complexes using α-conotoxin GIC, a well-known antagonistic ligand of nAChRs, and the extracellular domains of nAChRs that are homology modeled using the α1 subunit of mouse, which has much higher sequence homology with nAChR subunits than acetylcholine binding protein (AChBP).…”

“…Extensive structural characterization and comparison of several α-conotoxins suggested residues that might be important for their subtype recognition specificities Table 1 (14)(15)(16)(17)(18)(19)(20). In particular, the structural studies on the α4/7-conotoxins have shown that these toxins with a same "ω-shaped" backbone topology can display a widely different receptor recognition profiles by relying on subtle differences in their surface properties (14,15,19,21). Above all, the precise delineation of contact sites that are responsible for specific recognition between a particular receptor subtype and its ligands requires high-resolution structures of the complexes of many receptor subtypes with various ligands displaying a large window of affinities.…”

Solution Conformation of αA-conotoxin EIVA, a Potent Neuromuscular Nicotinic Acetylcholine Receptor Antagonist from Conus ermineus

“…obtain such information have not been successful (36 -38) even though a low resolution electromagnetic image (39,40) and an x-ray structure of an acetylcholine-binding protein that resembles the extracellular domain of nAChR have been obtained (41). In the absence of a high resolution structure of nAChR, we have been determining high resolution structures of ␣/␣A-conotoxins and tried to "reverse-predict" the receptor residues that might interact with the receptor-contacting residues of ligands (12)(13)(14)(15). Such an approach is possible because although various ␣/␣A-conotoxins exhibit a great variety of receptor subtype specificity, they differ only slightly in their sequences with a similar overall fold exhibiting subtle local structural differences.…”

“…In an effort to discover novel modulators of nAChR by identifying critical residues within the toxin for receptor contact, we have been applying a "reverse mapping strategy" using atomic resolution structures of various ␣/␣A-conotoxins (12)(13)(14)(15). Although considerable structure-function work has been reported on the ␣-conotoxins (16 -25), there has been only one report on the ␣A-subfamily (12) since their original discovery (6).…”