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Binding site of AChR: Difference between revisions

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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. <ref>PMID:18987633</ref> 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.<ref>PMID:18987633</ref> 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.<ref>PMID:18987633</ref>
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. <ref>PMID:18987633</ref> 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.<ref>PMID:18987633</ref> 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.<ref>PMID:18987633</ref>


The mechanism of pLGIC is provided by Prof. Jean-Pierre in the paper 'X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation'(Nature,2009): helices M1, M2 and M3, and a large portion of the β-sandwich consisting of strands b1, b2, b6, b7 and b10, these elements constitute the subunit ‘common core’. Common core superimposition shows that the GLIC subunits display a quaternary twist compared to ELIC, with anticlockwise (versus clockwise) rotation in the upper (versus lower) part of the pentamer, when viewed from the extracellular compartment (Fig. 3a). This is confirmed by normal mode analysis: the lowest frequency mode is precisely a twist mode and has by far the highest contribution (29%) to the transition. However, we note that the first 100 lowest-frequency modes (usually the most collective ones) only explain about 50% of the transition. Other 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 b-sandwich undergoes little deformation, but is rotated by 8u 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. The latter contains the well-conserved β6–β7 and M2–M3 loops and the b1–b2 loop whose length is conserved in the pLGIC family. We observe a downward motion of the β1–β2 loop, concomitant with a displacement of the M2–M3 loop, M2 and M3 helices and b6–b7 loop towards the periphery of the molecule (Fig. 3c), thereby opening the pore. Such twist to open motions, initially proposed from ab initio normal mode analysis of nAChRs, and observed for Kcsa, may plausibly be extended to eukaryotic pLGICs. 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.<ref>PMID:18987633</ref>
The mechanism of pLGIC is provided by Prof. Jean-Pierre in the paper 'X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation'(Nature,2009): helices M1, M2 and M3, and a large portion of the β-sandwich consisting of strands β1, β2, β6, β7 and β10, these elements constitute the subunit ‘common core’. Common core superimposition shows that the GLIC subunits display a quaternary twist compared to ELIC, with anticlockwise (versus clockwise) rotation in the upper (versus lower) part of the pentamer, when viewed from the extracellular compartment (Fig. 3a). This is confirmed by normal mode analysis: the lowest frequency mode is precisely a twist mode and has by far the highest contribution (29%) to the transition. However, we note that the first 100 lowest-frequency modes (usually the most collective ones) only explain about 50% of the transition. Other 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 b-sandwich undergoes little deformation, but is rotated by 8u 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. The latter contains the well-conserved β6–β7 and M2–M3 loops and the b1–b2 loop whose length is conserved in the pLGIC family. We observe a downward motion of the β1–β2 loop, concomitant with a displacement of the M2–M3 loop, M2 and M3 helices and b6–b7 loop towards the periphery of the molecule (Fig. 3c), thereby opening the pore. Such twist to open motions, initially proposed from ab initio normal mode analysis of nAChRs, and observed for Kcsa, may plausibly be extended to eukaryotic pLGICs. 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.<ref>PMID:18987633</ref>
   
   
== Superimpose HAP on AChBP ==
== Superimpose HAP on AChBP ==
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The ligand binding site of AChR is mainly located at the α-subunits. The acetylcholine binding protein(<scene name='68/688431/Achbp/2'>AChBP</scene>) is most closely related to the α-subunits of the nAChR. AChBP is a soluble protein found in the snail [http://en.wikipedia.org/wiki/Lymnaea_stagnalis Lymnaea stagnalis]. Nearly all residues that are conserved within the nAChR family are present in AChBP, including those that are relevant for lignad binding.<ref>PMID:11357122</ref> 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.<ref>PMID:11683996</ref>
The ligand binding site of AChR is mainly located at the α-subunits. The acetylcholine binding protein(<scene name='68/688431/Achbp/2'>AChBP</scene>) is most closely related to the α-subunits of the nAChR. AChBP is a soluble protein found in the snail [http://en.wikipedia.org/wiki/Lymnaea_stagnalis Lymnaea stagnalis]. Nearly all residues that are conserved within the nAChR family are present in AChBP, including those that are relevant for lignad binding.<ref>PMID:11357122</ref> 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.<ref>PMID:11683996</ref>


[[Image:Comparison between HAP and AChBP.PNG|thumb|350px|Fig. 3. Comparison, in Stere, of the 3D Structure of HAP(Red) and Loop 182-193 of AChBP(Blue)]]
[[Image:Comparison between HAP and AChBP.PNG|thumb|350px|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.
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.


[[Image:Combined BTX HAP and AchBP.png|thumb|350px|Fig. 4. A Stereo View of the Combined Model of α-BTX-HAP(Red) and AChBP subunits]]
[[Image:Combined BTX HAP and AchBP.png|thumb|350px|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 3), in the figure the red one is 13-mer little peptide and the blue one is loop 182-193 of AChBP.   
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 4 shows that the <scene name='68/688431/Btx_complex_with_two_subunits/1'>superposition of the HAP on loop 182-193</scene> 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: <scene name='68/688431/Btx_complex_with_two_subunits/5'>removing</scene> the HAP form the structure and <scene name='68/688431/Btx_complex_with_two_subunits/6'>
The figure 5 shows that the <scene name='68/688431/Btx_complex_with_two_subunits/1'>superposition of the HAP on loop 182-193</scene> 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: <scene name='68/688431/Btx_complex_with_two_subunits/5'>removing</scene> the HAP form the structure and <scene name='68/688431/Btx_complex_with_two_subunits/6'>
superposition</scene> the HAP on the AChBP.
superposition</scene> the HAP on the AChBP.


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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 [http://en.wikipedia.org/wiki/Cation%E2%80%93pi_interaction cation-π interactions], where the positive charge of the quaternary ammonium of acetylcholine interacts with the electron-rich aromatic side chains.<ref>PMID:11357122</ref> 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.<ref>PMID:11683996</ref> <scene name='68/688431/Hepes_five_subunits/2'>HEPES</scene> 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 <scene name='68/688431/Hepes_trp143/1'>Trp 143</scene> making cation-π interactions as expected for nicotinic agonists.<ref>PMID:11357122</ref>  
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 [http://en.wikipedia.org/wiki/Cation%E2%80%93pi_interaction cation-π interactions], where the positive charge of the quaternary ammonium of acetylcholine interacts with the electron-rich aromatic side chains.<ref>PMID:11357122</ref> 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.<ref>PMID:11683996</ref> <scene name='68/688431/Hepes_five_subunits/2'>HEPES</scene> 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 <scene name='68/688431/Hepes_trp143/1'>Trp 143</scene> making cation-π interactions as expected for nicotinic agonists.<ref>PMID:11357122</ref>  


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 <scene name='68/688431/Hepes_black_loop_2/1'>loop 2</scene> of α-BTX into that cleft. This clarifies and explains the strong inhibition of AChR function by the toxin.<ref>PMID:11683996</ref> The superposition of the HAP on loop 182-193 of AChBP(Fig.4)show that the major interaction between α-BTX and AChR α subunit, occur in residues187-192 of that sununit.   
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 <scene name='68/688431/Hepes_black_loop_2/1'>loop 2</scene> of α-BTX into that cleft. This clarifies and explains the strong inhibition of AChR function by the toxin.<ref>PMID:11683996</ref> 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.   


   
   

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Ma Zhuang, Zicheng Ye, Michal Harel, Angel Herraez, Alexander Berchansky
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