Binding site of AChR

Introduction

There are two kinds of acetylcholine receptor in nature: nicotinic acetylcholine receptors and muscarinic acetylcholine receptors. The nicotinic acetylcholine receptor(nAChR) is a pentameric ligand-gated ion channel activated by binding of acetylcholine in nature. In this page we will show the structure of binding site of nAChR by a complex between α-bungarotoxin and a mimotope peptide.

Basic structure of AChR

Pentameric ligand gated ion channels (pLGIC), or Cys-loop receptors,are a group of transmembrane ion channel proteins which open to allow ions such as Na+, K+, Ca2+, or Cl- to pass through the membrane in response to the binding of a chemical messenger, such as a neurotransmitter^[1]. In overall organization, the have five subunits. The five subunits are arranged in a barrel-like manner around a central symmetry axis that coincides with the ion permeation pathway.^[2] In each subunit, the extracellular domin(ECD) of pLGIC encompasses 10β-strands that are organized as a sandwich of two tightly interacting β-sheets, while the transmembrane domain(TMD) folds into a bundle of four α-helices (M1, M2, M3, M4).

Superimpose HAP on AChBP

There is a 13 amino acids high affinity peptide() which corresponding to residues 187-199 of the AChR that can inhibits the binding of α-BTX to AChR. And the high affinity and specific interaction of α-bungarotoxin () with AChR has been of considerable importance in the study of the binding site of AChR.^[3] So we want to use the complex between α-bungarotoxin and this little peptide to study the binding site of AChR.

The ligand binding site of AChR is mainly located at the α-subunits. The acetylcholine binding protein() is most closely related to the α-subunits of the nAChR. Nearly all residues that are conserved within the nAChR family are present in AChBP, including those that are relevant for lignad binding.^[4] And AChBP can also bind with α-Neurotoxins. So the AChBP structure is obviously an ideal candidate for testing the relevance of the conformation of the HAP when bound to α-BTX, to that of the corresponding binding region in AChR.^[5]

If we want to use the complex between α-bungarotoxin and the 13 amino acids to identify the binding site of the AChR, we should make sure that the little peptide has almost the same structure with the α-subunits of nAChR. From above we know that AChBP can be a model to study the structure of AChR. And the structure of AChBP has already been solved. So it easy to compare the little peptide with the AChBP.

Fig. 1. Comparison, in Stere, of the 3D Structure of HAP(Red) and Loop 182-193 of AChBP(Blue)

The overly of the first 12 residues of the 13-mer HAP on AChBP residues 182-193 shows that the HAP has almost the same conformation with the loop of AChBP(Fig 1), in the figure the red one is 13-mer little peptide and the blue one is a loop of AChBP. So it seems that the little peptide has almost the same structure with the region of AChR upon binding to α-BTX.

The figure blow shows that the the α-BTX to fit exquisitely into the interface of two subunits of the pentameric AChBP. it shows the stereo view of the combined model of α-BTX-HAP(Red) and AChBP structure with subunit A in green and subunit B in yellow showing the insertion of loop 2 of the toxin into the interface of the to subunits.In order to identify more clearly that the little 13-mer peptide is actually have almost the same structure with the 182-193 loop with AChBP, we compare two structures: and .

Image:Combined model of α-BTX-HAP and AchBP.png

It is noteworthy that the positively charged molecule shows the location of the acetylcholine binding site and the blockage of passage to this site caused by the toxin. So the ACh binding site in AChBP is assigned by the localization of HEPES.

Structure of Acetylcholine binding site

X-ray structure of homologues of the extracellular domain(ECD) of nAChRs have also been described:the acetylcholine binding protein(AChBP) co-crystallized with agonists and antagonists, and the ECD of α1-nAChRs.^[6]

The 13-mer HAP assumes an antiparallel β hairpin structure, and is held snugly between of α-BTX. The shortest and most numerous interactions are formed with finger 2 of α-BTX. The intermolecular interaction between finger2 and two arms of the HAP hairpin make the complex stable, like .

Affinity labeling experiments which indentified position 10 and 33 of -neurotoxin to be with in 11.5-15.5 Å from AChR residues Cys192-Cys193(Michalet et al ., 2000) agree with the α-BTX-HAP structure where the corresponding Cα distance are 11.05 Å(Pro10-Ser193), 12.72 Å(Cys33-Ser193), 14.45 Å（Pro10-Ser 192）,9.76 Å(Cys33-Ser192),respectively.^[7]So through the complex of α-BTX-HAP, we can see the structure of Acetylcholine binding site.

The superimposed model of AChBP and α-BTX suggests that the putative agonist HEPES seen in the AChBP structure is blocked from entering or leaving the AChBP interface cleft by the insertion of loop 2 of α-BTX into that cleft. This clarifies and explains the strong inhibition of AChR function by the toxin.^[8]

Function of Acetylcholine receptor

The α-Neurotoxins such as α-bungarotoxin (α-BTX)can compete antagonists of acetylcholine for its site. So studying the binding site of AChR is very important for the development of antidotesagainstα-BTX poisoning as well as drugs against, like Alzheimer's disease and nicotine addiction.

Nicotinic AChRs is neuron receptor protein that singal for muscular contraction upon the chemical stimulus.It may exist in different interconvertible conformational states. Binding of an agonist stabilises the open and desensitised states. Opening of the channel allows positively charged ions to move across it; in particular, sodium enters the cell and potassium exits. The net flow of positively-charged ions is inward.^[9]

The nAChR is unable to bind ACh when bound to any of the snake venom α-neurotoxins. These α-neurotoxins antagonistically bind tightly and noncovalently to nAChRs of skeletal muscles, thereby blocking the action of ACh at the postsynaptic membrane, inhibiting ion flow and leading to paralysis and death. The nAChR contains two binding sites for snake venom neurotoxins. Some studies have shown that a twist-like motion caused by ACh binding is likely responsible for pore opening, and that one or two molecules of α-bungarotoxin (or other long-chain α-neurotoxin) suffice to halt this motion. The toxins seem to lock together neighboring receptor subunits, inhibiting the twist and therefore, the opening motion.^[10]