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A Structural Model of the Human α7 Nicotinic Receptor in an Open Conformation.

Chiodo L, Malliavin TE, Maragliano L, Cottone G, Ciccotti G - PLoS ONE (2015)

Bottom Line: Homology modeling and Molecular Dynamics simulations are valuable tools to predict structures of unknown proteins, however, for the case of human nAchRs, they have been unsuccessful in providing a stable open structure so far.The channel remains stable and hydrated over the whole runs.Mostly based on the analysis of pore hydration and size, we suggest that the obtained structure has reasonable chances to be (at least one of the possible) structures of the channel in the open conformation.

View Article: PubMed Central - PubMed

Affiliation: Center for Life Nano Science @Sapienza, Istituto Italiano di Tecnologia, Rome, Italy.

ABSTRACT
Nicotinic acetylcholine receptors (nAchRs) are ligand-gated ion channels that regulate chemical transmission at the neuromuscular junction. Structural information is available at low resolution from open and closed forms of an eukaryotic receptor, and at high resolution from other members of the same structural family, two prokaryotic orthologs and an eukaryotic GluCl channel. Structures of human channels however are still lacking. Homology modeling and Molecular Dynamics simulations are valuable tools to predict structures of unknown proteins, however, for the case of human nAchRs, they have been unsuccessful in providing a stable open structure so far. This is due to different problems with the template structures: on one side the homology with prokaryotic species is too low, while on the other the open eukaryotic GluCl proved itself unstable in several MD studies and collapsed to a dehydrated, non-conductive conformation, even when bound to an agonist. Aim of this work is to obtain, by a mixing of state-of-the-art homology and simulation techniques, a plausible prediction of the structure (still unknown) of the open state of human α7 nAChR complexed with epibatidine, from which it is possible to start structural and functional test studies. To prevent channel closure we employ a restraint that keeps the transmembrane pore open, and obtain in this way a stable, hydrated conformation. To further validate this conformation, we run four long, unbiased simulations starting from configurations chosen at random along the restrained trajectory. The channel remains stable and hydrated over the whole runs. This allows to assess the stability of the putative open conformation over a cumulative time of 1 μs, 800 ns of which are of unbiased simulation. Mostly based on the analysis of pore hydration and size, we suggest that the obtained structure has reasonable chances to be (at least one of the possible) structures of the channel in the open conformation.

No MeSH data available.


Related in: MedlinePlus

The chimera.A,B) X-ray crystallographic structures of the chains A and E of AChBP from Aplysia californica (PDB entry: 2BYQ [26]), and GluCl from Caenorhabditis elegans (PDB entry: 3RIF, [41]), respectively. The structures are drawn in cartoons, and the regions used for superimposition are colored in red. The residues of the superimposed structures are: (i) 55–62, 77–80, 107–118, 91–93, 138–147 for 2BYQ, and (ii) 54–61, 76–79, 110–121, 91–93, 141–150 for 3RIF. C) Superimposed subunits from 2BYQ (green) and 3RIF (in blue) using the residues defined above. The Cys-loop regions are colored in blue for 2BYQ and in yellow for 3RIF, and the cysteines of the Cys-loop are drawn in magenta for 2BYQ and in red for 3RIF. The superimposition and figure were realized using pymol [70].
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pone.0133011.g002: The chimera.A,B) X-ray crystallographic structures of the chains A and E of AChBP from Aplysia californica (PDB entry: 2BYQ [26]), and GluCl from Caenorhabditis elegans (PDB entry: 3RIF, [41]), respectively. The structures are drawn in cartoons, and the regions used for superimposition are colored in red. The residues of the superimposed structures are: (i) 55–62, 77–80, 107–118, 91–93, 138–147 for 2BYQ, and (ii) 54–61, 76–79, 110–121, 91–93, 141–150 for 3RIF. C) Superimposed subunits from 2BYQ (green) and 3RIF (in blue) using the residues defined above. The Cys-loop regions are colored in blue for 2BYQ and in yellow for 3RIF, and the cysteines of the Cys-loop are drawn in magenta for 2BYQ and in red for 3RIF. The superimposition and figure were realized using pymol [70].

Mentions: As templates for homology modeling of the α7 nAChR we used the following X-ray structures: (i) AChBP from Aplysia californica bound to epibatidine (PDB entry: 2BYQ [26]), (ii) GluCl from Caenorhabditis elegans (PDB entry: 3RIF [41]). The two template structures have been manually handled to build a chimera protein in which the ECD domain (residues 0–203 in the chain A of 2BYQ) except the Cys-loop (residues 127–140 in the chain A of 2BYQ) was taken from AChBP, and the TMD domain (residues 209–339 in the chain A of 3RIF) as well as the Cys-loop (residues 130–144 in the chain A of 3RIF) were taken from GluCl. The motivation for this cutting of the structures is given in Fig 2, where the chains A of 3RIF and 2BYQ are superimposed. Indeed, in the 3RIF structure, the Cys-loop (in yellow on Fig 2C) interacts with the M2-M3 loop of TMD, whereas the corresponding region in 2BYQ (in blue on Fig 2C) moves apart from the M2-M3 loop. As the reciprocal position of the Cys-loop and M2-M3 loop is known to play an important role in the conformational transition of LGICs [53, 56–59] we decided to use the chimera protein described below as a template for the homology modeling. The homology model of α7 nAChR was then built using the chimera template and a sequence alignment of α7 and the chimera. The percentage of identity between the two sequences is 25.37%, similarly to what obtained in previous works using the same template [60, 61]. Models built from this degree of identity have a Ca RMSD of 2-3 Å with respect to the true structure [62, 63]. In these cases, a proper alignment of the sequences before building the model becomes extremely important. The software t_coffee 9.01 [64] was used to align the full GluCl sequence, the truncated GluCl sequence present in 3RIF, the α7 nAChR, the sequences of human α2, α3 and α4 of nAChR, and the chimera sequences, to determine the cytoplasmic region of α7. This region is thought to be folded as an α helix from the models determined using the EM map recorded on Torpedo acetylcholine receptor [9]. As this region has been replaced by a tripeptide in the GluCl sequence used for the determination of the 3RIF structure, we decided to replace the equivalent region of the α7 nAChR by the same tripeptide. We also truncated the N and C terminal extremities which contained many insertions in sequences alignment. The alignment of the truncated α7 nAChR sequence along the chimera sequence is described in Fig 3. Our alignment shows only 6 gaps, while functional regions such as the C-loop, the Cys-loop, as well as many conserved residues, are perfectly matched. Results are in full agreement with the sequence alignment between 2BYQ and the LBD of α7 realized by Grazioso et al. [61] (compare Fig 1 in [61] with Fig 3). The homology modeling was realized using the script build_model.py from Modeller 9.10 [65], with the refinement protocol very_slow and 10 refined models.


A Structural Model of the Human α7 Nicotinic Receptor in an Open Conformation.

Chiodo L, Malliavin TE, Maragliano L, Cottone G, Ciccotti G - PLoS ONE (2015)

The chimera.A,B) X-ray crystallographic structures of the chains A and E of AChBP from Aplysia californica (PDB entry: 2BYQ [26]), and GluCl from Caenorhabditis elegans (PDB entry: 3RIF, [41]), respectively. The structures are drawn in cartoons, and the regions used for superimposition are colored in red. The residues of the superimposed structures are: (i) 55–62, 77–80, 107–118, 91–93, 138–147 for 2BYQ, and (ii) 54–61, 76–79, 110–121, 91–93, 141–150 for 3RIF. C) Superimposed subunits from 2BYQ (green) and 3RIF (in blue) using the residues defined above. The Cys-loop regions are colored in blue for 2BYQ and in yellow for 3RIF, and the cysteines of the Cys-loop are drawn in magenta for 2BYQ and in red for 3RIF. The superimposition and figure were realized using pymol [70].
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4514475&req=5

pone.0133011.g002: The chimera.A,B) X-ray crystallographic structures of the chains A and E of AChBP from Aplysia californica (PDB entry: 2BYQ [26]), and GluCl from Caenorhabditis elegans (PDB entry: 3RIF, [41]), respectively. The structures are drawn in cartoons, and the regions used for superimposition are colored in red. The residues of the superimposed structures are: (i) 55–62, 77–80, 107–118, 91–93, 138–147 for 2BYQ, and (ii) 54–61, 76–79, 110–121, 91–93, 141–150 for 3RIF. C) Superimposed subunits from 2BYQ (green) and 3RIF (in blue) using the residues defined above. The Cys-loop regions are colored in blue for 2BYQ and in yellow for 3RIF, and the cysteines of the Cys-loop are drawn in magenta for 2BYQ and in red for 3RIF. The superimposition and figure were realized using pymol [70].
Mentions: As templates for homology modeling of the α7 nAChR we used the following X-ray structures: (i) AChBP from Aplysia californica bound to epibatidine (PDB entry: 2BYQ [26]), (ii) GluCl from Caenorhabditis elegans (PDB entry: 3RIF [41]). The two template structures have been manually handled to build a chimera protein in which the ECD domain (residues 0–203 in the chain A of 2BYQ) except the Cys-loop (residues 127–140 in the chain A of 2BYQ) was taken from AChBP, and the TMD domain (residues 209–339 in the chain A of 3RIF) as well as the Cys-loop (residues 130–144 in the chain A of 3RIF) were taken from GluCl. The motivation for this cutting of the structures is given in Fig 2, where the chains A of 3RIF and 2BYQ are superimposed. Indeed, in the 3RIF structure, the Cys-loop (in yellow on Fig 2C) interacts with the M2-M3 loop of TMD, whereas the corresponding region in 2BYQ (in blue on Fig 2C) moves apart from the M2-M3 loop. As the reciprocal position of the Cys-loop and M2-M3 loop is known to play an important role in the conformational transition of LGICs [53, 56–59] we decided to use the chimera protein described below as a template for the homology modeling. The homology model of α7 nAChR was then built using the chimera template and a sequence alignment of α7 and the chimera. The percentage of identity between the two sequences is 25.37%, similarly to what obtained in previous works using the same template [60, 61]. Models built from this degree of identity have a Ca RMSD of 2-3 Å with respect to the true structure [62, 63]. In these cases, a proper alignment of the sequences before building the model becomes extremely important. The software t_coffee 9.01 [64] was used to align the full GluCl sequence, the truncated GluCl sequence present in 3RIF, the α7 nAChR, the sequences of human α2, α3 and α4 of nAChR, and the chimera sequences, to determine the cytoplasmic region of α7. This region is thought to be folded as an α helix from the models determined using the EM map recorded on Torpedo acetylcholine receptor [9]. As this region has been replaced by a tripeptide in the GluCl sequence used for the determination of the 3RIF structure, we decided to replace the equivalent region of the α7 nAChR by the same tripeptide. We also truncated the N and C terminal extremities which contained many insertions in sequences alignment. The alignment of the truncated α7 nAChR sequence along the chimera sequence is described in Fig 3. Our alignment shows only 6 gaps, while functional regions such as the C-loop, the Cys-loop, as well as many conserved residues, are perfectly matched. Results are in full agreement with the sequence alignment between 2BYQ and the LBD of α7 realized by Grazioso et al. [61] (compare Fig 1 in [61] with Fig 3). The homology modeling was realized using the script build_model.py from Modeller 9.10 [65], with the refinement protocol very_slow and 10 refined models.

Bottom Line: Homology modeling and Molecular Dynamics simulations are valuable tools to predict structures of unknown proteins, however, for the case of human nAchRs, they have been unsuccessful in providing a stable open structure so far.The channel remains stable and hydrated over the whole runs.Mostly based on the analysis of pore hydration and size, we suggest that the obtained structure has reasonable chances to be (at least one of the possible) structures of the channel in the open conformation.

View Article: PubMed Central - PubMed

Affiliation: Center for Life Nano Science @Sapienza, Istituto Italiano di Tecnologia, Rome, Italy.

ABSTRACT
Nicotinic acetylcholine receptors (nAchRs) are ligand-gated ion channels that regulate chemical transmission at the neuromuscular junction. Structural information is available at low resolution from open and closed forms of an eukaryotic receptor, and at high resolution from other members of the same structural family, two prokaryotic orthologs and an eukaryotic GluCl channel. Structures of human channels however are still lacking. Homology modeling and Molecular Dynamics simulations are valuable tools to predict structures of unknown proteins, however, for the case of human nAchRs, they have been unsuccessful in providing a stable open structure so far. This is due to different problems with the template structures: on one side the homology with prokaryotic species is too low, while on the other the open eukaryotic GluCl proved itself unstable in several MD studies and collapsed to a dehydrated, non-conductive conformation, even when bound to an agonist. Aim of this work is to obtain, by a mixing of state-of-the-art homology and simulation techniques, a plausible prediction of the structure (still unknown) of the open state of human α7 nAChR complexed with epibatidine, from which it is possible to start structural and functional test studies. To prevent channel closure we employ a restraint that keeps the transmembrane pore open, and obtain in this way a stable, hydrated conformation. To further validate this conformation, we run four long, unbiased simulations starting from configurations chosen at random along the restrained trajectory. The channel remains stable and hydrated over the whole runs. This allows to assess the stability of the putative open conformation over a cumulative time of 1 μs, 800 ns of which are of unbiased simulation. Mostly based on the analysis of pore hydration and size, we suggest that the obtained structure has reasonable chances to be (at least one of the possible) structures of the channel in the open conformation.

No MeSH data available.


Related in: MedlinePlus