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Interactions of L-3,5,4'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

Westergard T, Salari R, Martin JV, Brannigan G - PLoS ONE (2015)

Bottom Line: T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces.Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites.In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America; Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, New Jersey, United States of America.

ABSTRACT
Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

No MeSH data available.


Poses indicated by automated docking of IVM (A), T3 (B), and ALLOP (C) to the GABAA receptor model.Five runs generating twenty poses each were conducted for all three ligands. Poses are colored according to docking score rank within a single run with strong scores in red, intermediate scores in white, and the weakest scores in blue, indicating that although poses for ALLOP and T3 are confined to subunit interfaces, multiple docking runs yield significant dispersion in orientation and ranking of individual interfaces (average scores in S1 Table). Poses located in the ion channel pore were excluded. The GABAA receptor transmembrane domain is shown and is colored by subunit: α-silver, β-green, γ-blue. In (B) and (C) the ligand hydroxyl is shown as a space-filling sphere to indicate orientation. Residues implicated by mutagensis for activation by ALLOP are shown in orange (34) and those photolabeled by etomidate (35) are shown in black.
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pone.0139072.g005: Poses indicated by automated docking of IVM (A), T3 (B), and ALLOP (C) to the GABAA receptor model.Five runs generating twenty poses each were conducted for all three ligands. Poses are colored according to docking score rank within a single run with strong scores in red, intermediate scores in white, and the weakest scores in blue, indicating that although poses for ALLOP and T3 are confined to subunit interfaces, multiple docking runs yield significant dispersion in orientation and ranking of individual interfaces (average scores in S1 Table). Poses located in the ion channel pore were excluded. The GABAA receptor transmembrane domain is shown and is colored by subunit: α-silver, β-green, γ-blue. In (B) and (C) the ligand hydroxyl is shown as a space-filling sphere to indicate orientation. Residues implicated by mutagensis for activation by ALLOP are shown in orange (34) and those photolabeled by etomidate (35) are shown in black.

Mentions: Docking calculations identified the subunit interface as a potential binding site for IVM, ALLOP, and T3 (Fig 5). Although these poses were likely facilitated by the cleft found in the GluCl template due to IVM binding in that structure, the asymmetry among docked poses suggests that the results are also sensitive to the receptor sequence. Asymmetry in docking scores (S1 Table) further suggests some degree of specificity, although the likely significance of these differences is reduced given dispersion in scores among identical sites (e.g. for ALLOP both the site with the least favorable and with the second most favorable average score were atβ- α interfaces). IVM and ALLOP were docked to all five subunits (each with a reduced average score for at least one of the β-α interfaces). T3 was only docked to the β-α and α- γ interfaces, with the latter having the most favorable score. This trend, however, was reversed in the MD simulations, as we discuss subsequently.


Interactions of L-3,5,4'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

Westergard T, Salari R, Martin JV, Brannigan G - PLoS ONE (2015)

Poses indicated by automated docking of IVM (A), T3 (B), and ALLOP (C) to the GABAA receptor model.Five runs generating twenty poses each were conducted for all three ligands. Poses are colored according to docking score rank within a single run with strong scores in red, intermediate scores in white, and the weakest scores in blue, indicating that although poses for ALLOP and T3 are confined to subunit interfaces, multiple docking runs yield significant dispersion in orientation and ranking of individual interfaces (average scores in S1 Table). Poses located in the ion channel pore were excluded. The GABAA receptor transmembrane domain is shown and is colored by subunit: α-silver, β-green, γ-blue. In (B) and (C) the ligand hydroxyl is shown as a space-filling sphere to indicate orientation. Residues implicated by mutagensis for activation by ALLOP are shown in orange (34) and those photolabeled by etomidate (35) are shown in black.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589331&req=5

pone.0139072.g005: Poses indicated by automated docking of IVM (A), T3 (B), and ALLOP (C) to the GABAA receptor model.Five runs generating twenty poses each were conducted for all three ligands. Poses are colored according to docking score rank within a single run with strong scores in red, intermediate scores in white, and the weakest scores in blue, indicating that although poses for ALLOP and T3 are confined to subunit interfaces, multiple docking runs yield significant dispersion in orientation and ranking of individual interfaces (average scores in S1 Table). Poses located in the ion channel pore were excluded. The GABAA receptor transmembrane domain is shown and is colored by subunit: α-silver, β-green, γ-blue. In (B) and (C) the ligand hydroxyl is shown as a space-filling sphere to indicate orientation. Residues implicated by mutagensis for activation by ALLOP are shown in orange (34) and those photolabeled by etomidate (35) are shown in black.
Mentions: Docking calculations identified the subunit interface as a potential binding site for IVM, ALLOP, and T3 (Fig 5). Although these poses were likely facilitated by the cleft found in the GluCl template due to IVM binding in that structure, the asymmetry among docked poses suggests that the results are also sensitive to the receptor sequence. Asymmetry in docking scores (S1 Table) further suggests some degree of specificity, although the likely significance of these differences is reduced given dispersion in scores among identical sites (e.g. for ALLOP both the site with the least favorable and with the second most favorable average score were atβ- α interfaces). IVM and ALLOP were docked to all five subunits (each with a reduced average score for at least one of the β-α interfaces). T3 was only docked to the β-α and α- γ interfaces, with the latter having the most favorable score. This trend, however, was reversed in the MD simulations, as we discuss subsequently.

Bottom Line: T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces.Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites.In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America; Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, New Jersey, United States of America.

ABSTRACT
Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

No MeSH data available.