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Binding site of AChR

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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).

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.[3]

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.[4] The little peptide can bind to α-BTX as competitive inhibitors of α-BTX biding to AChR. So the complex between α-BTX and this little peptide(HAP) maybe can used as a model 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. AChBP is a soluble protein found in the snail Lymnaea stagnalis. Nearly all residues that are conserved within the nAChR family are present in AChBP, including those that are relevant for lignad binding.[5] 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.[6]

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

In order to use the complex between α-BTX and HAP to identify the binding site of the AChR, the structure of HAP should homologous with the α-subunits of nAChR. AChBP is a very important and ideal model to study the structure of AChR, which structure has already been solved. So comparing the HAP with the AChBP will show whether HAP can be used as a model to study the binding site of AChR.

Fig. 2. A Stereo View of the Combined Model of α-BTX-HAP(Red) and AChBP subunits

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 182-193 of AChBP(Fig 1), in the figure the red one is 13-mer little peptide and the blue one is loop 182-193 of AChBP.

The figure 2 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. The blue little peptide is HAP, which superimpose on the loop 182-193 of AChBP. 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: the HAP form the structure and the HAP on the AChBP.

So that the little peptide(HAP) has almost the same structure with the loop of AChBP binding to α-BTX, which means it can be used as a model to study the binding site of AChR.

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

In nAChR, the ligand-binding site is located at the interface between two subunits. The homopentameric α7 receptor contains five identical ligand binding sites. In these sites acrtylcholine is expected to bind through cation-π interactions, where the positive charge of the quaternary ammonium of acetylcholine interacts with the electron-rich aromatic side chains.[7] can be refined in the current AChBP structure, it does not make any specific hydrogen bonds with the protein, it stacks with its quaternary ammonium onto making cation-π interactions as expected for nicotinic agonists.[8] 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 of α-BTX into that cleft. This clarifies and explains the strong inhibition of AChR function by the toxin.[9]

The 13-mer assumes an antiparallel β hairpin structure. It is held snugly between of α-BTX. The shortest and most numerous interactions are formed with of α-BTX. The two arms of the HAP hairpin assume a β sheet conformation, with residues Leu2 (corresponding to position 188 in AChR)-Tyr4 (corresponding to position 190 in AChR ) making an with α-BTX residues Val39-Glu41 on a loop region. Tyr3 (corresponding to position 189 in AChr) of HAP forms a sung fit into a loop region of α-BTX. The formation of from its hydroxyl to residues Thr8 and lle11 of α-BTX makes the tyrosine at that position an ideal candidate for forming binding interactions with α-BTX. Indeed, this tyrosine is known to play a crucial role in α-BTX binding. [10]



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.[11]

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.[12]


structure of binding site of AChR

Drag the structure with the mouse to rotate




QuizQuiz

1 nAChR is...?

Dimeric ligand-gated ion channel
Trimeric ligand-gated ion channel
Tetramer ligand-gated ion channel
Pentameric ligand-gated ion channel

2 How many residues HAP has?

11
12
13
14

3 HAP is a part of AChBP

True
False

4 What will happen when αBTX binding to AChR?

The channel will open
The subunits will be locked
Nothing will happen

5 Which finger of αBTX has the shortest and most numerous interaction with HAP?

1
2
3
4


ReferencesReferences

  1. ↑ Purves, Dale, George J. Augustine, David Fitzpatrick, William C. Hall, Anthony-Samuel LaMantia, James O. McNamara, and Leonard E. White (2008). Neuroscience. 4th ed. Sinauer Associates. pp. 156–7. ISBN 978-0-87893-697-7.
  2. ↑ Gonzalez-Gutierrez G, Cuello LG, Nair SK, Grosman C. Gating of the proton-gated ion channel from Gloeobacter violaceus at pH 4 as revealed by X-ray crystallography. Proc Natl Acad Sci U S A. 2013 Oct 28. PMID:24167270 doi:http://dx.doi.org/10.1073/pnas.1313156110
  3. ↑ Bocquet N, Nury H, Baaden M, Le Poupon C, Changeux JP, Delarue M, Corringer PJ. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature. 2009 Jan 1;457(7225):111-4. Epub 2008 Nov 5. PMID:18987633 doi:10.1038/nature07462
  4. ↑ Harel M, Kasher R, Nicolas A, Guss JM, Balass M, Fridkin M, Smit AB, Brejc K, Sixma TK, Katchalski-Katzir E, Sussman JL, Fuchs S. The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide. Neuron. 2001 Oct 25;32(2):265-75. PMID:11683996
  5. ↑ Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, Sixma TK. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature. 2001 May 17;411(6835):269-76. PMID:11357122 doi:10.1038/35077011
  6. ↑ Harel M, Kasher R, Nicolas A, Guss JM, Balass M, Fridkin M, Smit AB, Brejc K, Sixma TK, Katchalski-Katzir E, Sussman JL, Fuchs S. The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide. Neuron. 2001 Oct 25;32(2):265-75. PMID:11683996
  7. ↑ Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, Sixma TK. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature. 2001 May 17;411(6835):269-76. PMID:11357122 doi:10.1038/35077011
  8. ↑ Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van Der Oost J, Smit AB, Sixma TK. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature. 2001 May 17;411(6835):269-76. PMID:11357122 doi:10.1038/35077011
  9. ↑ Harel M, Kasher R, Nicolas A, Guss JM, Balass M, Fridkin M, Smit AB, Brejc K, Sixma TK, Katchalski-Katzir E, Sussman JL, Fuchs S. The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide. Neuron. 2001 Oct 25;32(2):265-75. PMID:11683996
  10. ↑ Harel M, Kasher R, Nicolas A, Guss JM, Balass M, Fridkin M, Smit AB, Brejc K, Sixma TK, Katchalski-Katzir E, Sussman JL, Fuchs S. The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide. Neuron. 2001 Oct 25;32(2):265-75. PMID:11683996
  11. ↑ http://en.wikipedia.org/wiki/Nicotinic_acetylcholine_receptor
  12. ↑ Samson AO, Levitt M. Inhibition mechanism of the acetylcholine receptor by alpha-neurotoxins as revealed by normal-mode dynamics. Biochemistry. 2008 Apr 1;47(13):4065-70. doi: 10.1021/bi702272j. Epub 2008 Mar 8. PMID:18327915 doi:http://dx.doi.org/10.1021/bi702272j

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