Structural evidence for the binding of monocarboxylates and dicarboxylates at pharmacologically relevant extracellular sites of a pentameric ligand-gated ion channel

Abstract

<jats:p>GLIC is a bacterial homologue of the pentameric ligand-gated ion channels (pLGICs) that mediate the fast chemical neurotransmission of nerve signalling in eukaryotes. Because the activation and allosteric modulation features are conserved among prokaryotic and eukaryotic pLGICs, GLIC is commonly used as a model to study the allosteric transition and structural pharmacology of pLGICs. It has previously been shown that GLIC is inhibited by some carboxylic acid derivatives. Here, experimental evidence for carboxylate binding to GLIC is provided by solving its X-ray structures with a series of monocarboxylate and dicarboxylate derivatives, and two carboxylate-binding sites are described: (i) the `intersubunit' site that partially overlaps the canonical pLGIC orthosteric site and (ii) the `intrasubunit' vestibular site, which is only occupied by a subset of the described derivatives. While the intersubunit site is widely conserved in all pLGICs, the intrasubunit site is only conserved in cationic eukaryotic pLGICs. This study sheds light on the importance of these two extracellular modulation sites as potential drug targets in pLGICs.</jats:p>

Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations

“…As a model system in this family, the Gloeobacter violaceus proton-gated ion channel (GLIC) has historically offered both insights and limitations ( 7 ). This prokaryotic receptor has been functionally characterized in multiple cell types ( 8 ) and crystallizes readily under activating conditions (pH ≤ 5.5) ( 9 , 10 ), producing apparent open structures up to 2.22 Å resolution ( 11 ) in the absence and presence of various ligands ( 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ) and mutations ( 22 , 23 , 24 , 25 ). Additional low-pH X-ray structures of GLIC have been reported in lipid-modulated ( 26 ) and the so-called locally closed states ( 27 , 28 , 29 , 30 ), with a hydrophobic constriction at the pore midpoint (I233, I9′ in prime notation) as predicted for closed channels throughout the family ( 31 ).…”

Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations

“…As a model system in this family, the Gloeobacter violaceus proton-gated ion channel (GLIC) has historically offered both insights and limitations [8] . This prokaryotic receptor has been functionally characterized in multiple cell types [9] and crystallizes readily under activating conditions (pH ≤ 5.5) [10], [11] , producing apparent open structures up to 2.22 Å resolution [12] in the absence and presence of various ligands [13]- [22] and mutations [23]- [26] . Additional low-pH X-ray structures of GLIC have been reported in lipid-modulated [27] and so-called locally closed states [28]- [31] , with a hydrophobic constriction at the pore midpoint (I233, I9' in prime notation) as predicted for closed channels throughout the family [32] .…”

Short-chain di-carboxylates as positive allosteric modulators of the pH-dependent pentameric ligand-gated ion channel GLIC: requirement of an intact vestibular pocket

“…In docking predictions in GLIC, both CAFFE (Prevost et al 2013) and CROTON (Alqazzaz et al 2016) were located slightly lower than the pLGICs reference site. And the inter-SU site slightly deeper location was confirmed in all available CBX-bound GLIC structures (Fourati et al 2020). As a consequence, the inter-SU pocket in GLIC is accessible from the periphery of the pentamer through an empty peripheral entrance, which in Eukaryotes is part of the orthotopic site.…”

“…In the context of a single occupancy, one hypothesis (among two) is that binding occurs only at the inter-SU pocket, as neurotransmitters bind to the orthotopic site in Eukaryote receptors. Involvement of the inter-SU pocket as the major di-CBX binding site is supported by crystallographic data from GLIC-di-CBX cocrystals, showing that FUMAR and SUCCIN are both found occupying the inter-SU pocket in their respective co-crystals (Fourati et al 2020). The region corresponding to the vestibular pocket would then condition inter-SU/orthotopic binding itself, or/and the transmission of forces between the orthotopic site and the rest of the protein during allosteric transitions, determining the gating probabilities.…”

“…Indeed, Arg77 side chain is oriented towards the vestibular pocket (occupied by a chloride ion) in the apo-GLIC structure obtained using phosphate as pH buffer (PDB reference 4qh5; Fourati et al 2015). Whereas, in all the CBX-bound GLIC structures available, it is found coordinating the CBX-molecule bound into the inter-SU pocket, whatever is the status of the vestibular pocket (occupied by a CBX molecule or occupied by a chloride ion/'CBX-empty') (Sauguet et al 2013, Fourati et al 2020. This includes four structures of GLIC in complex with molecules of the di-CBX compounds analyzed in the present study, malonate (6hjb), succinate (6hjz), glutarate (6hja) and fumarate (6hj3).…”