Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4–α4 interface

Ahring, Philip K.; Olsen, Jeppe; Nielsen, Elsebet Ø.; Peters, Dan; Pedersen, Martin Holst Friborg; Rohde, L.A.H.; Kastrup, J.S.; Shahsavar, Azadeh; Indurthi, Dinesh C.; Chebib, Mary; Gajhede, Michael; Balle, Thomas

doi:10.1016/j.neuropharm.2014.12.035

Cited by 21 publications

(29 citation statements)

References 33 publications

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“…4 A and B). These positions have been shown to be important for the diverse ligand specificity seen in HS and LS subtypes of α4β2 nAChR (37,38). Superposition of loops B and C of the α2-Epi structure with the corresponding loops of the epibatidine-bound structures of α7-AChBP chimera (18) and AcAChBP (13) reveals a lateral rotation of epibatidine toward Tyr226 by ∼8° (Fig.…”

Section: Resultsmentioning

confidence: 96%

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

Kouvatsos

Giastas

Chroni-Tzartou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(−) binding site on the heteromeric low sensitivity α2β2 nAChR and validated the functional importance of specific residues in α2 and β2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structurebased design of subtype specific drugs against the nAChR associated diseases.cys-loop receptors | α2β2 nAChR | X-ray crystallography | ligand-gated ion channel

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Section: Resultsmentioning

confidence: 96%

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

Kouvatsos

Giastas

Chroni-Tzartou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…4 G and H, Right). Notably, previous studies show that expressing the heteromeric α4β2 nAChR under conditions that favor an (α4β2) 2 α4 stoichiometry (three α4 and two β2 subunits) results in a receptor having two α4(+)/β2(−) interfaces with high agonist sensitivity and a third binding site at the α4(+)/α4(−) interface that displays low agonist sensitivity (59)(60)(61).…”

Section: Discussionmentioning

confidence: 99%

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

Degani-Katzav

Gortler

Gorodetzki

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the “principal” and “complementary” components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits’ cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

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“…Crystals of both complexes were obtained using the hanging drop In the presented fits bottom was set to 0 and the Hill slope to 1. Data for WT a4b2 and a4b2 HQT are from Ahring et al, 2015. vapor diffusion method at 20°C. Crystallization drops were made by mixing 1 ml of a 5 mg/ml protein:ligand solution with 1 ml of crystallization solution containing 0.1 M Tris-HCl buffer (pH 8.0), 1-3% v/v polyethylene glycol 400, and 1.8-2.3 M (NH 4 ) 2 SO 4 .…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we have shown that three residues, H142, Q150, and T152 on the complementary side of the a4 subunit (a4(2)) and V136, F144, and L146 on the corresponding side of b2 (b2(2)), comprise the difference between the core of a-a and a-b binding sites, respectively, and are accountable for differences in agonist sensitivities (Harpsøe et al, 2011) and agonist binding affinities between the two sites (Ahring et al, 2015). Further, we have shown that a modulator that selectively targets the a-a binding site may substitute for one of the agonist molecules to aid activation via an agonist-like mechanism (Olsen et al, 2014a).…”

Section: Introductionmentioning

confidence: 99%

“…We introduced the three a-a site "signature" residues in Ls-AChBP (Ls-AChBP HQT ) and conducted radioligand-binding experiments. The derived ligand affinities were then correlated to a-a affinities obtained from a functional a4b2 receptor in which a-b sites were engineered to resemble a-a sites by mutation of the three signature residues in the b2 subunit (a4b2 HQT ) to ease detection of a-a binding (Ahring et al, 2015). Next, we determined crystal structures of complexes between Ls-AChBP HQT and the a4b2 agonists NS3920 [1-(6-bromopyridin-3-yl)-1,4-diazepane] and NS3573 [1-(5-ethoxypyridin-3-yl)-1,4-diazepane] (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

Shahsavar¹,

Ahring²,

Olsen³

et al. 2015

Mol Pharmacol

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Neuronal a4b2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two a4 subunits in the (a4) 3 (b2) 2 receptor subpopulation was discovered. In particular, three residues, H142, Q150, and T152, were demonstrated to be involved in the distinct pharmacology of the a4-a4 versus a4-b2 binding sites. To obtain insight into the three-dimensional structure of the a4-a4 binding site, a surrogate protein reproducing a4-a4 binding characteristics was constructed by introduction of three point mutations, R104H, L112Q, and M114T, into the binding pocket of Lymnaea stagnalis acetylcholine-binding protein (Ls-AChBP). Cocrystallization with two agonists possessing distinct pharmacologic profiles, NS3920 [1-(6-bromopyridin-3-yl)-1,4-diazepane] and NS3573 [1-(5-ethoxypyridin-3-yl)-1,4-diazepane], highlights the roles of the three residues in determining binding affinities and functional properties of ligands at the a4-a4 interface. Confirmed by mutational studies, our structures suggest a unique ligandspecific role of residue H142 on the a4 subunit. In the cocrystal structure of the mutated Ls-AChBP with the high-efficacy ligand NS3920, the corresponding histidine forms an intersubunit bridge that reinforces the ligand-mediated interactions between subunits. The structures further reveal that the binding site residues gain different and ligand-dependent interactions that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting specific nicotinic acetylcholine receptor interfaces.

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Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4–α4 interface

Cited by 21 publications

References 33 publications

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

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