Short-chain mono-carboxylates as negative modulators of allosteric transitions in GLIC, and impact of a pre-β5 strand (Loop Ω) double mutation on crotonate, not butyrate effect

Abstract

Using the bacterial proton-activated receptor-channel GLIC, we identify a locus in the pre-β5 strand (Loop Ω) whose mutation inverses the effect of the mono-carboxylate crotonate from negative to positive modulation of the allosteric transitions, suggesting an involvement of the pre-β5 strand in coupling the extracellular orthotopic receptor to pore gating in this pentameric ligand-gated ion channel. As an extension to the previously proposed ″in series″ mechanism, we suggest that a orthotopic/orthosteric site — vestibular site — Loop Ω — β5-β6 ″sandwich″ — Pro-Loop/Cys-Loop series may be an essential component of orthotopic/orthosteric compound-elicited gating control in pentameric ligand-gated ion channels, on top of which compounds targeting the vestibular site may provide modulation.

Fumarate as positive modulator of allosteric transitions in the pentameric ligand‐gated ion channel GLIC: requirement of an intact vestibular pocket

“…In an associated report (see preliminary data in Van Renterghem et al., Preprint), we establish a different, ‘loose’ pattern of impact of the same CBX‐pocket single mutations, on mono‐CBXs NAM effects. From the ‘loose’ pattern of mutational impact, and published crystallographic data, and from the properties of a pre‐β5 (Loop Ω) double mutant, while keeping the ‘in series’ mechanism, we propose that, in addition to being a coupling region, the vestibular pocket may function as an accessory, allotopic binding site, exerting a veto between the main orthotopic binding site and the gating machinery.…”

“…Unexpectedly, N152A impact was an increased fumar PAM effect (preliminary data). As a consistent explanation appeared in comparing mono‐CBX and di‐CBX N152A data, the di‐CBX and caffe data for N152A are presented and discussed with mono‐CBX data in a follow‐on report (see preliminary data in Van Renterghem et al., Preprint). All other constructs presented a pHo 50 within the inclusion criterion (±0.5 pH unit from the wt value; i.e.…”

“…Caffeate is an orthotopic NAM. In an associated report (see preliminary data in Van Renterghem et al., Preprint), we look for a contribution of allotopic binding (binding at the intra‐SU pocket) to PAM or NAM effects. The proposed terminology has the advantage of not using the same word (‘allosteric’) to designate two different concepts (both present in Monod, Wyman & Changeux, 1965): other binding site locations, and conformational transitions between active/inactive states.…”

Discovering cryptic pocket opening and binding of a stimulant derivative in a vestibular site of the 5-HT3Areceptor

“…1C) [7], and various modulatory carboxylates in the proton-gated channel GLIC (Fig. S1) [8][9][10]. The channel sTeLIC, derived from an endosymbiont of the tubeworm Tevnia jerichonana, was recently crystallized with the positive modulator 4-bromocinnamate in its vestibule [11] (Fig.…”

“…Similarly, in the plant-pathogen channel ELIC and proton-gated channel GLIC, the benzodiazepine flurazepam (PDB ID: 2YOE, chain C) [9] and various modulatory carboxylates (PDB IDs: 6HJZ, 4QH1, 6HPP), respectively, were found to bind in this location (Fig. S1) [10][11][12]. In these cases, ligands were identified between the β4, β5, and β6 strands, in a vestibular cavity defined by the so-called Ω-loop (Fig.…”