[[Image:Mechanism of GLIC.PNG|thumb|350px|Fig. 3. Open GLIC and closed ELIC structure comarison green is GLIC and red is ELIC]]
[[Image:Mechanism of GLIC.PNG|thumb|350px|Fig. 3. Open GLIC and closed ELIC structure comarison green is GLIC and red is ELIC]]
The general mechanism of pLGIC is provided by Prof. Jean-Pierre Changeux in the paper 'X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation[J]. Nature, 2009, 457(7225): 111-114.'. GLIC and ELIC are both pentameric ligand gated ion channel which in the same family with AChR and AChBP. GLIC is an apparently open conformation while ELICis presumed closed conformation. Comparative analysis of GLIC and ELIC reveals the rotation of β-sandwich and a tilt of M2 and M3, which will show the mechanism of pLGIC. Common core is consists by M1, M2 and M3, and a large portion of the β-sandwich. The superimposition of common core shows that the GLIC subunits display an anticlock quaternary twist compared to ELIC(Fig. 3a).
The general mechanism of pLGIC is provided by Prof. Jean-Pierre Changeux in the paper 'X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation[J]. Nature, 2009, 457(7225): 111-114'. GLIC and ELIC are both pentameric ligand gated ion channel which in the same family with AChR and AChBP. GLIC is an apparently open conformation while ELICis presumed closed conformation. Comparative analysis of GLIC and ELIC reveals the rotation of β-sandwich and a tilt of M2 and M3, which will show the mechanism of pLGIC. Common core is consists by M1, M2 and M3, and a large portion of the β-sandwich. The superimposition of common core shows that the GLIC subunits display an anticlock quaternary twist compared to ELIC(Fig. 3a).
But in the first 100 lowest-frequency modes only 50% of the transition can be explained. The rest and more local movements occur: in the TMD the outer ends of M2 and M3 of GLIC are tilted away radially from the channel axis, while the outer end of M1 is fixed. The inner ends of M1, M2 and M3 move tangentially towards the left, when viewed from the membrane (Fig. 3b). In the ECD, the core of the β-sandwich undergoes little deformation, but is rotated by 8° around an axis roughly perpendicular to the inner sheet of the β-sandwich (Fig. 3a), concomitant with a rearrangement of both the subunit–subunit and the ECD/TMD interfaces, regions known to contribute to neurotransmitter gating. A downward motion of the β1–β2 loop, concomitant with a displacement of the M2–M3 loop, M2 and M3 helices and β6–β7 loop towards the periphery of the molecule (Fig. 3c), thereby opening the pore<ref name="Bocquet2009" />.
But in the first 100 lowest-frequency modes only 50% of the transition can be explained. The rest and more local movements occur: in the TMD the outer ends of M2 and M3 of GLIC are tilted away radially from the channel axis, while the outer end of M1 is fixed. The inner ends of M1, M2 and M3 move tangentially towards the left, when viewed from the membrane (Fig. 3b). In the ECD, the core of the β-sandwich undergoes little deformation, but is rotated by 8° around an axis roughly perpendicular to the inner sheet of the β-sandwich (Fig. 3a), concomitant with a rearrangement of both the subunit–subunit and the ECD/TMD interfaces, regions known to contribute to neurotransmitter gating. A downward motion of the β1–β2 loop, concomitant with a displacement of the M2–M3 loop, M2 and M3 helices and β6–β7 loop towards the periphery of the molecule (Fig. 3c), thereby opening the pore<ref name="Bocquet2009" />.
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 and opening mechanism of AChR
Fig. 1. Structure of GLIC
Acetylcholine receptor is a member of pentameric ligand gated ion channels family,which share the similar structure. 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 pLGICs 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).
Fig. 2. Top view of GLIC M2 helices
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. Most pLGICs undergo desensitization on prolonged exposure to agonist, complicating structural investigations of the transient open conformation[3]. The overall architecture of bacterial Gloeobacter violaceus pentameric ligand-gated ion(GLIC) is similar to nAChR(Fig 1). The five subunits are arranged in a barrel-like manner around a central symmetry axis that coincides with the ion permeation pathway[3]. The transmembrane domain of each subunit consists of four helices and M2 helices form the wall of the pore(Fig 2).Figure 2 shows that helix backbones and side chains facing the pore are depicted. Hydrophobic, polar and negative residues are coloured yellow, green and red respectively. The M2 axes are tilted with respect to the pore axis, with outer hydrophobic side chain oriented toward the helix interfaces, and inner polar side chains oriented towards the pore[3].
Fig. 3. Open GLIC and closed ELIC structure comarison green is GLIC and red is ELIC
The general mechanism of pLGIC is provided by Prof. Jean-Pierre Changeux in the paper 'X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation[J]. Nature, 2009, 457(7225): 111-114'. GLIC and ELIC are both pentameric ligand gated ion channel which in the same family with AChR and AChBP. GLIC is an apparently open conformation while ELICis presumed closed conformation. Comparative analysis of GLIC and ELIC reveals the rotation of β-sandwich and a tilt of M2 and M3, which will show the mechanism of pLGIC. Common core is consists by M1, M2 and M3, and a large portion of the β-sandwich. The superimposition of common core shows that the GLIC subunits display an anticlock quaternary twist compared to ELIC(Fig. 3a).
But in the first 100 lowest-frequency modes only 50% of the transition can be explained. The rest and more local movements occur: in the TMD the outer ends of M2 and M3 of GLIC are tilted away radially from the channel axis, while the outer end of M1 is fixed. The inner ends of M1, M2 and M3 move tangentially towards the left, when viewed from the membrane (Fig. 3b). In the ECD, the core of the β-sandwich undergoes little deformation, but is rotated by 8° around an axis roughly perpendicular to the inner sheet of the β-sandwich (Fig. 3a), concomitant with a rearrangement of both the subunit–subunit and the ECD/TMD interfaces, regions known to contribute to neurotransmitter gating. A downward motion of the β1–β2 loop, concomitant with a displacement of the M2–M3 loop, M2 and M3 helices and β6–β7 loop towards the periphery of the molecule (Fig. 3c), thereby opening the pore[3].
At the beginning, this kind of twist to open motions is come from ab initio normal mode analysis of nAChRs, and then plausibly be extended to the pLGICs family. The structural transition described here couples in an allosteric manner the opening–closing motion of the pore with distant binding sites—located at the ECD subunit interface for neurotransmitters, or within the TMD for allosteric effectors30—and may possibly serve as a general mechanism of signal transduction in pLGICs[3].
Superimpose HAP on AChBP
There is a 13 amino acids high affinity peptide(HAP) 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 (α-BTX) 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(AChBP) 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[4].
Fig. 4. 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. 5. 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 4), in the figure the red one is 13-mer little peptide and the blue one is loop 182-193 of AChBP.
The figure 5 shows that the superposition of the HAP on loop 182-193 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: removing the HAP form the structure and
superposition 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 HEPES 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
The 13-mer HAP assumes an antiparallel β hairpin structure, which can be used as a model to study the binding site of AChR. It is held snugly between fingers 1,2 and 4 of α-BTX. The shortest and most numerous interactions are formed with finger 2 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 intermolecular interaction 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 two H bonds 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[4].
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[5]. The ACh binding site in AChBP was assigned by the localization of a solvent molecule (positively charge HEPES) seen near residues corresponding to the 187-199 loop of the AChR α subunit and stacking on the corresponding Trp 143[4]. HEPES 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 Trp 143 making cation-π interactions as expected for nicotinic agonists[5].
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[4]. The superposition of the HAP on loop 182-193 of AChBP(Fig.5)show that the major interaction between α-BTX and AChR α subunit, occur in residues187-192 of that sununit.
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[6].
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[7].
↑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.
↑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:https://dx.doi.org/10.1073/pnas.1313156110
↑ 3.03.13.23.33.4Bocquet 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
↑ 4.04.14.24.34.4Harel 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
↑ 5.05.15.2Brejc 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
↑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:https://dx.doi.org/10.1021/bi702272j
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