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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

Ions distribution.Blue bars: cations, that can permeate the nicotinic channel. Red bars: anions. Top panels show the channel pore profiles along the full-length protein. Central panel: restrained; lower panel: unrestrained trajectory. The cyan region including the cyan rectangle in the top panel) highlights the channel between Glu20′ and Leu9′. The vertical black lines on the pore profile highlight the cytoplasmic and the extracellular pore limits, from Gly2′ to Glu20′.
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pone.0133011.g010: Ions distribution.Blue bars: cations, that can permeate the nicotinic channel. Red bars: anions. Top panels show the channel pore profiles along the full-length protein. Central panel: restrained; lower panel: unrestrained trajectory. The cyan region including the cyan rectangle in the top panel) highlights the channel between Glu20′ and Leu9′. The vertical black lines on the pore profile highlight the cytoplasmic and the extracellular pore limits, from Gly2′ to Glu20′.

Mentions: The α7 nAChR is cation selective. To further assess the α7 model proposed in this study, we performed an analysis of the distribution of both cations and anions in the channel pore. Fig 10 shows the distribution of the z-coordinate of ions along the restrained and unrestrained trajectories, from the intracellular to the extracellular exits, both for the sodium (cations) and chlorine (anions) ions. Ion populations calculated along the free trajectories are shown in S9 Fig. The overall profile along the full-length channel is in agreement with results reported in literature for nicotinic receptors, though obtained with higher ions concentration and/or via the application of a transmembrane potential along Brownian Dynamics simulations (see e.g Fig 7 in Ref. [89]). Starting from the bottom of the protein (-30 Å), the anions populate more the cytoplasmic extremity of the channel (-30 Å<z<-20 Å), but the cations could enter more into the channel, in the portion comprised between -20Å and 0 Å; this in particular in the unrestrained trajectory and, on average, along the four free trajectories. The hydrophobic gate, in the range 0 Å<z<20 Å is not populated. Indeed, given the low, physiological salt concentration (100 mM) used here to neutralize the total system charge, and, most importantly, the time scale of our MD simulation (200ns for each trajectory), it is highly improbable to detect any conductive event crossing the hydrophobic gate, where the free energy barrier to ion permeation is located. However, despite the low concentration here used with respect to other studies devoted to ion motion [89, 90], along one of the free trajectories (Free4) we observed the simultaneous presence of two cations at the same time in the region just below the Leu9′ hydrophobic ring (-10 Å<z<0Å) [91]. This is a precursor event for the ion passage through the hydrophobic gate [89]. Ions population in the LBD shows an alternation of anions and cations. We observe in particular a cation excess located at z ∼ 30Å and z ∼ 50Å, in correspondence to charged residue rings composed by Asp38, Glu39 and Asp91, Glu92 respectively. These rings corresponds to the ones identified in Ref. [90] to be associated with prolonged cation dwell time.


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)

Ions distribution.Blue bars: cations, that can permeate the nicotinic channel. Red bars: anions. Top panels show the channel pore profiles along the full-length protein. Central panel: restrained; lower panel: unrestrained trajectory. The cyan region including the cyan rectangle in the top panel) highlights the channel between Glu20′ and Leu9′. The vertical black lines on the pore profile highlight the cytoplasmic and the extracellular pore limits, from Gly2′ to Glu20′.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133011.g010: Ions distribution.Blue bars: cations, that can permeate the nicotinic channel. Red bars: anions. Top panels show the channel pore profiles along the full-length protein. Central panel: restrained; lower panel: unrestrained trajectory. The cyan region including the cyan rectangle in the top panel) highlights the channel between Glu20′ and Leu9′. The vertical black lines on the pore profile highlight the cytoplasmic and the extracellular pore limits, from Gly2′ to Glu20′.
Mentions: The α7 nAChR is cation selective. To further assess the α7 model proposed in this study, we performed an analysis of the distribution of both cations and anions in the channel pore. Fig 10 shows the distribution of the z-coordinate of ions along the restrained and unrestrained trajectories, from the intracellular to the extracellular exits, both for the sodium (cations) and chlorine (anions) ions. Ion populations calculated along the free trajectories are shown in S9 Fig. The overall profile along the full-length channel is in agreement with results reported in literature for nicotinic receptors, though obtained with higher ions concentration and/or via the application of a transmembrane potential along Brownian Dynamics simulations (see e.g Fig 7 in Ref. [89]). Starting from the bottom of the protein (-30 Å), the anions populate more the cytoplasmic extremity of the channel (-30 Å<z<-20 Å), but the cations could enter more into the channel, in the portion comprised between -20Å and 0 Å; this in particular in the unrestrained trajectory and, on average, along the four free trajectories. The hydrophobic gate, in the range 0 Å<z<20 Å is not populated. Indeed, given the low, physiological salt concentration (100 mM) used here to neutralize the total system charge, and, most importantly, the time scale of our MD simulation (200ns for each trajectory), it is highly improbable to detect any conductive event crossing the hydrophobic gate, where the free energy barrier to ion permeation is located. However, despite the low concentration here used with respect to other studies devoted to ion motion [89, 90], along one of the free trajectories (Free4) we observed the simultaneous presence of two cations at the same time in the region just below the Leu9′ hydrophobic ring (-10 Å<z<0Å) [91]. This is a precursor event for the ion passage through the hydrophobic gate [89]. Ions population in the LBD shows an alternation of anions and cations. We observe in particular a cation excess located at z ∼ 30Å and z ∼ 50Å, in correspondence to charged residue rings composed by Asp38, Glu39 and Asp91, Glu92 respectively. These rings corresponds to the ones identified in Ref. [90] to be associated with prolonged cation dwell time.

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