Unravelling the Conformational Plasticity of the Extracellular Domain of a Prokaryotic nAChR Homologue in Solution by NMR

Chasapis, Christos T.; Argyriou, Aikaterini I.; Corringer, Pierre‐Jean; Bentrop, Detlef; Spyroulias, Georgios A.

doi:10.1021/bi201223u

Cited by 4 publications

(5 citation statements)

References 10 publications

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“…Each of these subunits is composed of a N-terminal extracellular ligand-binding domain (ECD) and an ionotropic transmembrane domain (TMD). In addition to integral pLGICs, several isolated ECD and TMD structures have been solved: the water-soluble pentameric AChBP homolog (9), the ECD of α1 nAChR in complex with α-bungarotoxin (10), α7nAChR/AChBP chimeras (11,12), GLIC ECD (13,14), as well as isolated TMD of the α1nAChR (15) and α1GlyR (16). Nevertheless, the functional implications concerning ligandactivation and modulation that can be deduced from isolated domains or monomeric structures are limited because the vast majority of allosteric modulatory sites are located at the interface between adjacent subunits or close to the ECD/TMD interface.…”

Section: Overall Structure: Conservation Of a Common Corementioning

confidence: 99%

Crystallographic studies of pharmacological sites in pentameric ligand-gated ion channels

Sauguet

Shahsavar

Delarue

2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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Section: Overall Structure: Conservation Of a Common Corementioning

confidence: 99%

Crystallographic studies of pharmacological sites in pentameric ligand-gated ion channels

Sauguet

Shahsavar

Delarue

2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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Section: Discussionmentioning

confidence: 99%

“…Previous attempts have been made to express isotopically-labelled ligand binding domains of nAChR homologs to monitor conformational changes in solution [ 35 , 37 ]. However, these are limited by the absence of an intact ligand binding pocket as is the case for gleobacter violaceus ligand-gated ion channel which remains a monomer in solution [ 35 ]. AChBP overcomes this limitation and not only exists as a pentamer in solution, but has been shown to possess channel gating properties, therefore providing a reliable template to observe ligand-associated channel gating mechanisms [ 52 ].…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, attempts have been made to use NMR techniques to monitor ligand induced conformational changes of the nAChR in solution. Isotopically labelled ( 15 N and 13 C) ligand binding domains of bacterial homologs have been expressed for this purpose, but these failed to oligomerize as a pentamer in solution and therefore lacked an intact ligand binding pocket [ 35 , 36 ]. AChBP naturally assembles as a pentamer in solution and therefore is a suitable candidate for such studies.…”

Section: Introductionmentioning

confidence: 99%

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Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

et al. 2016

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Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

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“…In contrast, quaternary structural assembly of an isolated ECD is less certain. The isolated ECD of GLIC presents as a hexamer in crystal structure [70] or monomer in solution NMR [71], even though its tertiary structure remains the same as that observed in the pentameric structure of the full-length GLIC [30, 31]. Varying oligomerization states were also observed for the ECD α1-GlyR subunit [72].…”

Section: Use Of Nmr For Structures and Dynamics Of Cys-loop Receptorsmentioning

confidence: 99%

Atomistic insights into human Cys-loop receptors by solution NMR

Mowrey

Kinde

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) mediating fast neurotransmission in the central and peripheral nervous systems. They are important targets for many currently used clinical drugs, such as general anesthetics, and for allosteric modulators with potential therapeutic applications. Here, we provide an overview of advances in the use of solution NMR in structural and dynamic characterization of ion channels, particularly human Cys-loop receptors. We present challenges to overcome and realistic solutions for achieving high-resolution structural information for this family of receptors. We discuss how subtle structural differences among homologous channels define unique channel pharmacological properties and advocate the necessity to determine high-resolution structures for individual receptor subtypes. Finally, we describe drug binding to the Cys-loop receptors’ TMD identified by solution NMR and the associated dynamics changes relevant to channel functions.

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Unravelling the Conformational Plasticity of the Extracellular Domain of a Prokaryotic nAChR Homologue in Solution by NMR

Cited by 4 publications

References 10 publications

Crystallographic studies of pharmacological sites in pentameric ligand-gated ion channels

Crystallographic studies of pharmacological sites in pentameric ligand-gated ion channels

Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

Atomistic insights into human Cys-loop receptors by solution NMR

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