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• W4384074620 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the cryo-EM structure of the lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators. Editor's evaluation The authors solved cryoEM structural maps for the pLGIC homolog Alpo4 from an extreme thermophile worm in apo and CHAPS bound conditions. The data are convincing and valuable and reveal how a detergent can bind to the orthosteric site and induce a quaternary twist of the channel domains. A limitation is that it is difficult to relate these observations to channel function as the activating ligand for Alpo4 remains unknown. https://doi.org/10.7554/eLife.86029.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Cys-loop receptors are allosterically regulated pentameric ligand-gated ion channels (pLGICs). Functionally pLGICs are classified into cation- and anion-selective receptors. The former class is exemplified by nicotinic acetylcholine (nAChRs) and serotonin (5-HT3) receptors (Corringer et al., 2012). Anion-selective receptors are comprised of glycine (GlyRs) and GABAA receptors. Because of their role in synaptic transmission, the inflammatory response and implication in diseases including gastro-intestinal, psychiatric and cognitive disorders, startle disease, epilepsy, and smoking addiction (Treiman, 2001; Albuquerque et al., 2009; Walstab et al., 2010; Ha and Richman, 2015), human and mammalian homologs of pLGICs have been the subject of active research. Recent advances in cryo-EM led to a rapid expansion of the structural data on nAChRs. The structures of three types of heteropentameric nicotinic receptors (α4β2, α3β4, and Torpedo muscle-nAChR) and one homopentameric receptor (α7) were determined (Morales-Perez et al., 2016; Walsh et al., 2018; Gharpure et al., 2019; Rahman et al., 2022; Noviello et al., 2021). For the α7 nicotinic receptor, structures of three major conformational states were solved: a resting, an agonist-bound activated, and an agonist-bound desensitized state. Cryo-EM structures also have been determined for 5-HT3 receptors (Basak et al., 2018; Polovinkin et al., 2018; Hassaine et al., 2014, glycine receptors Huang et al., 2015; Du et al., 2015), and GABAA receptors (Kim et al., 2020; Masiulis et al., 2019; Zhu et al., 2018; Laverty et al., 2019; Miller and Aricescu, 2014). Currently, only one structure of a non-vertebrate ion channel, the glutamate-gated chloride channel (GluCl), from C. elegans has been solved. GluCl is the drug target for anthelmintics such as ivermectin highlighting the importance of structural characterization of non-vertebrate pLGICs (Althoff et al., 2014; Hibbs and Gouaux, 2011). Lophotrochozoa comprises one of the largest groups in the animal kingdom and includes organisms such as annelids, mollusks, and platyhelminths (flatworms). Intriguingly, genome analysis revealed a massive expansion of nAChR genes in these organisms with 52 and 217 nAChR genes identified in mollusks and annelids, respectively (Jiao et al., 2019). This contrasts with the number of receptors found in organisms having an advanced nervous system, in which only 10–20 nAChRs are encoded in the genomes, for example, 17 in humans (Walsh et al., 2018). It has been speculated that the expansion in nAChR genes is a consequence of the adaptation to a stationary lifestyle in a dynamic environment (Jiao et al., 2019). The biological role of the additional nAChRs and their properties remain unknown. Characterization of these receptors may lead to discoveries of alternative neurotransmitters, new signaling pathways as well as a better understanding of the evolution of neurotransmission. We have previously biochemically characterized seven invertebrate Cys-loop receptors, Alpo1-7, identified in the proteome of Alvinella pompejana, an annelid worm that inhabits the surroundings of hydrothermal vents and is the most extreme thermophilic invertebrate currently known (Chevaldonné et al., 2000; Holder et al., 2013). Among seven Alpo receptors, we identified two nAChR-like receptors (Alpo1 and Alpo4) and one Gly-like receptor (Alpo6), which were expressed and purified in amounts suitable for structural studies (Wijckmans et al., 2016). Alpo4 has 27–29% sequence identity with α-subunits of nAChRs and 25% with 5-HT3 receptors. The high biochemical stability and preliminary characterization using negative stain electron microscopy suggested that Alpo4 was a promising target for structural studies (Kocot et al., 2017). However, despite exhaustive screening in different expression systems in combination with a compound library, including acetylcholine and serotonin, we could not identify the agonist/neurotransmitter for Alpo4, thereby limiting functional studies (Wijckmans et al., 2016). Because of its biochemical stability and unique position between nAChRs and 5-HT3 receptors, we characterized the structure of Alpo4 using single-particle electron cryogenic microscopy (cryo-EM). Results Alpo4 is an isolated member of lophotrochozoan nAChRs A massive expansion of nAChR genes in lophotrochozoans suggests their importance in functional diversity and adaptations. In A. pompejana the total number of nAChR genes is not known because its genome has not been fully sequenced. To get further insight into the relation of Alpo4 to other nAChRs we applied comparative genomic analysis. We performed a phylogenetic comparison of Alpo1-4 with nAChRs from annelids: Capitella teleta (CT), Dimorphilus gyrociliatus (DM), Owenia fusiformis (OW), Hirudo verbana (HV), Helobdella robusta (HR), and from mollusca: Crassostrea virginca (MV), Crassostrea gigas (CG), Mizuhopecten yessoensis (MY), Pecten maximus (PM) and Pomacea canaliculata (PC) (Kocot et al., 2017). A total of 649 sequences were grouped into 25 families (Figure 1—figure supplement 1a). In the phylogenetic tree some sequences cluster in molluscan-specific families (e.g. 58A, 58B, 61A, 62A, and 63A), whereas other families, like 33A, include members from all lophotrochozoan and have characteristic features of a vertebrate α7 subunit (Li et al., 2016). Alpo1-4 were classified into different sequence families. Alpo2 and 3 are found in the families 32C and 41A (sequence identities of the closest homolog are 45% and 65%, respectively), both of which contain sequences from the genome of each included lophotrochozoan suggesting functional importance and conservation of these protein families within the clade (Figure 1—figure supplement 1b). Interestingly, in Polychaeta organisms, the characteristic vicinal disulfide in the tip of loop C required for ligand binding is not present in Alpo3-like nAChRs. On the contrary, Alpo1 and Alpo4 (families 42A and 43A; sequence identities of the closest homolog 36% and 37%) do not clusters in the well-populated families (Figure 1—figure supplement 1b). This suggests either a unique function or a faster evolution. To further characterize Alpo4, we proceeded with its structural characterization by cryo-EM. Structure of Alpo4 reveals CHAPS bound to the orthosteric site We purified Alpo4 in the detergent LMNG following the protocol established earlier (Wijckmans et al., 2016) and used cryo-EM to solve its structure. The map reconstructed to 4.1 Å resolution confirmed that Alpo4 assembles into a homopentamer and has a conserved architecture of the pLGIC family (Figure 1a, Table 1). Each Alpo4 subunit is composed of a β-sandwich extracellular domain (ECD) and a transmembrane domain (TMD) made of four trans-membrane α-helices M1-M4 (Figure 1b, Figure 1—figure supplement 2b). Helices M2 contributed by each subunit are radially arranged around a central ion-conducting pore. The density for the intracellular domain (ICD), residues 308–412, was missing, and it was consequently not modeled (Figure 1—figure supplement 2b). An additional density next to the side chain of N167 on ECD was modeled as an N-acetylglucosamine (GlcNAc; Figure 1b, Figure 1—figure supplement 2b). Glutamine glycosylation at the structurally equivalent position was also found in the 5-HT3 receptor, but not in nicotinic receptors (Polovinkin et al., 2018). Figure 1 with 10 supplements see all Download asset Open asset Overview of solved Alpo4 structures. (a) Electron cryogenic microscopy (Cryo-EM) reconstruction of apo Alpo4CHAPS (blue), Alpo4APO (green), Alpo4ACH (purple), Alpo4APO_LMNG (orange), and Alpo4SER (yellow). The detergent micelle is shown in white surface representation. A monomer is shown in a darker shade. The density corresponding to bound CHAPS is shown in violet. (b) Side view of the atomic models shown in cartoon representation with NAG moieties shown as sticks. One subunit is highlighted. Bound CHAPS molecules are shown as sticks (violet). Table 1 Statistics of cryo-EM data collection, data processing, and model refinement. Data depositionAlpo4 ID:Alpo4CHAPSAlpo4APOAlpo4APO_LMNGAlpo4ACHAlpo4COMB*Alpo4SERPDB ID:8BYI8BXF8BX58BXB8BXE8BXDEMDB ID:EMD-16326EMD-16317EMD-16308EMD-16314EMD-16316EMD-16315Data collectionMicroscopeJOEL CRYOARM300Acceleration voltage [kV]300Energy filterIn-column Omega energy filterEnergy filter slit width [eV]20Spherical aberration [mm]2.55Magnification60 000DetectorGatan K2Gatan K3Gatan K3Gatan K3Gatan K3Gatan K3Refined pixel size [Å]0.7820.7840.75960.75960.75960.7596Exposure time [s]2.9853.9552.985 /3.9553.955Number of frames616159595959Electron exposure [e-/Å2]3730455945/5959Defocus range [μm]1.6–2.80.8–2.81.0–2.41.0–2.41.0–2.41.0–2.4Collected images5003715311 83913 83025 6692200Used images268237549387781613 6471595Particles picked372 518939 3701 242 2872 597 373508 966446 221Data processingSymmetryC5C5C5C5C5C5Particles refined23 54318 654135 177251 656131 38079 454Final resolution [Å],FSC = 0.1434.13.44.23.93.96.2Sharpening B-factor [Å2]–176–139–297–170–235–1062Local resolution range [Å]3.7–4.62.9–5.03.5–7.73.2–5.83.2–5.85.5–7.0Model refinementRefinement packagePHENIX 1.19Initial model used6HIQAlpo4CHAPSAlpo4ACHAlpo4APOAlpo4ACHAlpo4ACHModel resolution [Å], FSC = 0.54.23.94.54.24.1Model composition Non-hydrogen protein atoms13 32112 69212 55012 54712 553 Protein residues16001630163016301600 Ligands13131313131B-factors mean [Å2] Protein63932035493 Ligand4010819954111R.M.S deviations Bond lengths (Å)0.0020.0050.0030.0040.004 Bond angles (°)0.6881.2400.7360.7791.055Validation Molprobity score1.91.61.82.31.9 Clashscore24.14.814.17.212.5 Poor rotamers (%)00.405.50Ramachandran plot Favored (%)9896979695 Allowed (%)24345 Disallowed (%)00000 * Combined Alpo4ACH and Alpo4APO. Although no ligand was added, an additional density was observed in the orthosteric binding site located at the interface between the ECDs (Figure 1a, Figure 1—figure supplement 4a). Despite the limited resolution, this density was well-resolved and consistent with a CHAPS molecule, a steroid-derived detergent. We further refer to this structure as Alpo4CHAPS. CHAPS was present in the purified Alpo4 at a concentration of 0.007% (110 μM) because of its thermostabilizing effect on Alpo4 (Wijckmans et al., 2016). At 110 μM, the CHAPS concentration is 70-fold lower than the critical micellar concentration (CMC) of the detergent, suggesting a specific interaction with Alpo4 beyond modulating properties of the detergent belt and, therefore, is consistent with CHAPS binding to the orthosteric site (Petroff et al., 2022). Structure of the ECD and ligand-binding pocket with CHAPS The ECD comprises an amino-terminal α-helix followed by 10 β-strands folded into a β-sandwich (Figure 1b, Figure 1—figure supplement 2b). CHAPS is bound at the ligand-binding pocket located at the interface of the principal (loops A-C) and the complementary subunit (loops D-F; Figure 2a and d). The CHAPS-Alpo4 interactions can be divided into two regions: the hydrophilic moiety and the sterol-binding moiety (Figure 2—figure supplement 1a). The hydrophilic moiety, in part formed by the dimethylammonio group, shares structural resemblance with carbachol and overlaps with the canonical Cys-loop receptor ligand-binding site, involving a group of highly conserved aromatic residues F103 (loop A), W159 (loop B), Y199, and Y205 (loop C) of the principal subunit and W65 (Loop D) of the complimentary subunit. Here, the quaternary ammonium group of the CHAPS molecule establishes a cation-π interaction with W159 (Figure 2a and d). This interaction is strikingly similar to the cation-π interactions observed with the quaternary ammonium group of the carbachol-bound AChBP structure (PDB: 1UV6), or the pyrrolidine nitrogen group in the nicotine-bound α4β2 nAChR structure (PDB: 5KXI) (Figure 2b and e). The interaction with the hydrophilic moiety is further stabilized by a salt bridge between the sulfonate group of CHAPS and Alpo4-specific Arg171 (Figure 2a). Figure 2 with 2 supplements see all Download asset Open asset Orthosteric binding site of Alpo4. (a, d) Ligand binding pocket of Alpo4 with bound CHAPS. The residues interacting with CHAPS are shown as sticks. (b, e) Similar views of the ligand binding site in the acetylcholine binding protein (AChBP) in complex with carbachol (PDB code 1UV6). (c, f) Ligand binding pocket of Alpo4 in apo state. Residues constituting the aromatic cage are shown as sticks. The self-liganded state of W159 is shown as an inset in sticks representation. The principal subunit is colored in a lighter shade and complimentary in a darker. The sterol ring moiety of CHAPS is composed of three cyclohexane and one cyclopentane ring and it fits into a hydrophobic crevice with a high shape-complementarity. The sterol ring interacts with residues F103 (loop A), N104, F137, V155, and W197 on the principal side and residues V48, K49, and D181 on the complementary side exclusively via Van der Waals contacts (Figure 2a, Figure 2—figure supplement 1a). This hydrophobic crevice is specific to Alpo4 and it is lined by poorly conserved residues. In other nAChRs, the pocket is narrower (Figure 2—figure supplement 1b) and is lined by multiple charged residues. These molecular interactions of CHAPS with Alpo4 explain why the binding of the detergent is specific. Structure of the ligand-binding pocket in the apo state To gain further insight into the structural and ligand-binding properties of Alpo4, several structures were solved in the absence of CHAPS (Figure 1). This was accomplished in two ways. Either CHAPS was removed from the Alpo4CHAPS using size exclusion chromatography or we purified Alpo4 without any CHAPS present (Table 1). The highest resolution reconstruction of 3.4 Å was obtained from the protein sample that was depleted of CHAPS using size-exclusion chromatography (Figure 1—figure supplement 4). This reconstruction, which we refer to as the apo state (Alpo4APO), allowed detailed modeling of the atomic structure of Alpo4 in most parts of the density (Figure 1—figure supplement 5). Despite the higher overall resolution of the reconstruction, the density corresponding to the tip of the C- and the F- loops was less well resolved which indicates their increased flexibility in the absence of a ligand. At low sigma levels, residual densities were observed in the ligand-binding and the sterol-binding pockets suggesting that residual CHAPS was bound at low occupancy. Similar results were obtained from the sample prepared in LMNG without CHAPS, reconstructed to 4.1 Å (Figure 1—figure supplement 6). This structure is essentially identical to the apo state with (RMSD of 0.9 Å) (Figure 1—figure supplement 7). No residual density was observed in the ligand-binding pocket in the later reconstruction, supporting the assignment of the 3.4 Å map as an apo state. The conformation of the aromatic residues constituting the ligand-binding pocket differed from the CHAPS-bound state. Specifically, W159 (loop B) flips and forms a cation-π interaction with H131, while being pinched by W65 (loop D) (Figure 2f). In this conformation, W159 can no longer form a cation-π interaction with an external ligand, therefore, it is tempting to speculate that this conformation represents a ‘self-liganded’ state. Given that Alpo4 shares a structural resemblance to the α4β2 nAChR and the 5-HT3R, we investigated whether acetylcholine or serotonin binds into the ligand-binding site. To this end, structures of Alpo4 purified in the absence of CHAPS and with an added, 1 mM acetylcholine or 1 mM serotonin were solved to a resolution of 3.9 Å and 6.2 Å, respectively (Figure 1, Figure 1—figure supplements 8 and 10). Their overall conformation was identical to that of the apo state with an overall RMSD of 0.87 and 0.88 Å, respectively. No density in the ligand-binding site was observed in the reconstructions. Although 6.2 Å resolution is too low to interpret the density of serotonin, the overall quaternary structure was identical to that of the apo state. The absence of a quaternary twist expected for a desensitized conformation suggests that serotonin was not bound. The cryo-EM reconstructions of Alpo4 obtained in the presence of acetylcholine and serotonin suggest that neither of the neurotransmitters binds Alpo4. This agrees with our electrophysiological experiments in various expression systems (Xenopus oocytes, HEK cells, lipid vesicles) indicating no agonist response to acetylcholine or serotonin (data not shown). Conformational changes upon binding of CHAPS A comparison of the CHAPS-bound structure with the apo state reveals concerted conformational changes. In addition to the local rearrangements of side chains in the ligand-binding site (described above), we observe a clear change in the quaternary conformation of Alpo4 (Figure 3, Video 1). Upon binding of CHAPS, the ECD rotates 9° clockwise relative to the TMD (when viewed from the extracellular side; Figure 3a and b). The binding of CHAPS is associated with local conformational changes in the ECD. First, the tip of the loop C (residues 197–205) shifts by about 3 Å and extends (Figure 3a and d) even though its density in the apo state is somewhat ambiguous (Figure 1—figure supplement 5). The loop movement is accompanied by changes in the orientation of aromatic sidechains (Y195, W197, and Y199) that allow accommodating the zwitterionic moiety of the CHAPS molecule (Figures 2 and 3d; Video 1). On the complimentary subunit, loop F (residues 171–184) shifts by 3–5 Å toward the sterol group of CHAPS. This results in a small (~1 Å) rearrangement of the ECD-TMD linker (Figure 3a and e). The ECD protomers show concerted movements as rigid bodies. They rotate by ~3 degrees around the domain center such that the apical regions of ECD move in the direction of neighboring ECD subunit in a clockwise fashion, whereases TMD-facing ends move counterclockwise (Figure 3a, Video 1). The whole ECD assembly rearranges as tightly packed domino tiles. The quaternary rearrangements preserve the hydrophobic CHAPS-binding groove (Figure 3f and g) even though its width changes. Figure 3 with 1 supplement see all Download asset Open asset Conformational changes between apo and CHAPS-bound Alpo4. (a) Structures of apo (green) and CHAPS-bound (light blue) Alpo4 are overlayed. The extracellular domain (ECD) was aligned between the structures. Bound CHAPS is shown in pink for reference. Only one subunit from each pentamer is colored, others are shown in gray for clarity. The binding of CHAPS results in an around 3 degrees clockwise rotation. The approximate rotation center is indicated by the black dot. The rotation of the membrane domain is indicated by an arrow. (b) Relative rotation of transmembrane domain (TMD) and ECD. The structures are aligned to TMDs. (c) Same as panel (b) but only TMDs are shown. Changes in TMD associated with CHAPS binding are minor. (d, e) Close-up of conformational changes in loops C and F, respectively. (f, g) Surface electrostatics is shown around sterol-binding grooves for apo (f) and CHAPS-bound states (g). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Transition of Alpo4APO to Alpo4CHAPS viewed from the extracellular side, side view, the C-loop, and the F-loop. We can speculate that the combination of CHAPS-induced rigid body tilt of the individual ECDs relative to each other and local rearrangements in the F loop leads to the rotation of TMD relative to ECD. This rotation is accommodated by the bending of the first two helical turns of the M1 helix, between 1 and 3 Å, an extension of the FPF motif on the Cys-loop, and a 3–5 Å shift of M2-M3 loop that follows rigid body movement rotation of the Cys-loop (Figure 3a and c; Video 1). A quaternary twist is associated with gating transitions in characterized pLGICs (Noviello et al., 2021; Basak et al., 2018; Polovinkin et al., 2018; Zarkadas et al., 2022; Petroff et al., 2022; Sauguet et al., 2014; Kumar et al., 2020). Quaternary changes in Alpo4 induced upon CHAPS binding and those associated with the activation of related α7 nACh and 5-HT3 receptors induced rotation of ECD relative to TMD in the same direction, however, the shifts of principal relative to complementary subunits were different (Video 2). In Alpo4, the complementary subunit slides upward whereas in the two other channels, it consistently shifts towards the principal subunit and tilts relative to the TMD. The tilt is less pronounced in Alpo4 which is probably why it does not lead to the pore dilation. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Quaternary conformational changes in Alpo4 upon binding of CHAPS are shown along with quaternary changes in α7 nicotinic acetylcholine (nACh) and 5-HT3 receptors upon transition from resting to active state. The channels were aligned to the extracellular surface of the pore to show rotation of extracellular domain (ECD) relative to transmembrane domain (TMD) and to ECD of one subunit to show relative movements of ECDs. One subunit is shown in purple. Structure of the pore domain In the TMD, all four TM helices are well resolved allowing for unambiguous assignment of the helix register. The densities for the M1-M3 helices are of excellent quality whereas the peripheral M4 displayed higher mobility (Figure 1—figure supplement 5). The ion pore is located along the fivefold rotational symmetry axis and is formed exclusively by the M2 helices. It has a circa 15 Å long hydrophobic patch in the outer leaflet of the membrane formed by three helical turns (residues 9’L, 13’L, and 16’M; Figure 4a). On the extracellular side, the hydrophobic region is flanked by negatively charged aspartate residues, 20’D, whereas on the intracellular side glycines G’6 create a cavity within the pore (Figure 4b) followed by rings of threonines (2’T) and conserved glutamates (–1’E) which usually play a role of the selectivity filter in cation-selective pLGICs (Yonekura et al., 2015). Thus, the charge distribution along the pore is consistent with Alpo4 being selective for cations. Figure 4 Download asset Open asset Permeation pathway of Alpo4. (a) Pore diameter calculated using HOLE and represented as dots for Alpo4CHAPS (blue) and Alpo4APO (green). Only M2 is shown in the cartoon and the pore-facing residues are shown as sticks. Constrictions are shown in red. (b) Pore diameter along the channel axis for Alpo4CHAPS (blue), Alpo4APO (green), and α4β2 (gray, PDB: 5KXI). The zero value along the channel axis corresponds to position 2’ (Thr251). Figure 4—source data 1 The pore diameter of the channels shown in panel b is calculated by HOLE. https://cdn.elifesciences.org/articles/86029/elife-86029-fig4-data1-v2.xlsx Download elife-86029-fig4-data1-v2.xlsx A highly unusual feature in Alpo4 is the presence of bulky M265 residues at the 16’ position. It forms the narrowest and most hydrophobic constriction (diameter of 2.1 Å) that likely functions as a gate. Constriction at this position is absent in other structurally characterized nAChRs but was observed in the bacterial Cys-loop receptor homolog ELIC in which the pore is constricted by a Phe residue at the extracellular surface (Ulens et al., 2014). This observation led us to speculate that the narrow 16’ constriction prevented ion permeation, leading to a lack of agonist responses in our earlier electrophysiological ligand screenings. Therefore, we explored an M16’L mutation, which unfortunately was also unresponsive to acetylcholine or serotonin (data not shown). Next, the M16’L mutation was combined with the well-described L9’T mutation, which slows desensitization, converts certain antagonists into agonists, and increases Ca2+ permeability in α7 nAChRs (Galzi et al., 1992). In our experiments, the double mutant M16’L/L9’T was still unresponsive to acetylcholine or serotonin. Additionally, we constructed chimeras in which the ECD and TMD were swapped with the α7 nAChR, similar to the α7/5-HT3 chimera (Eiselé et al., 1993), but these constructs also remained unresponsive. Finally, we also considered that the 16’ methionine residues could confer redox-sensitive channel regulation, similar to the upper gate formed in TRPV2 ion channels (Fricke et al., 2019). However, we could not detect any channel activity in the presence of oxidizing (H2O2) or reducing agents (DTT) (data not shown). In conclusion, Alpo4 has a pore structure consistent with a cation-selective channel, but with an unusually tight constriction at the 16’M position, the role of which remains unclear. In silico ligand screening To deorphanize Alpo4, we performed virtual screening using 37,000 compounds on three conformations of the Alpo4 receptor (see Methods for details). For compounds that were identified across multiple simulations, binding energies were calculated and ranked accordingly to select hit compounds with the greatest likelihood of producing agonistic activity. After examining the docking poses of the hits, only compounds docked within the ligand binding cavity were retained (Figure 2—figure supplement 2 and Supplementary file 2). Several of the top compounds contain sterol-like moieties including the top hit from virtual screening, ZINC36126889, which has an average binding energy of –12.1 kcal/mol (Supplementary file 2). This is a natural product synthesized by various species of Solanum plants and contains the sapogenin backbone structure. Metagenin also contains the sapogenin backbone, however, is more hydroxylated than ZINC36126889 and has an average binding energy of –11.3 kcal/mol. These compounds are structurally similar to diosgenin – a bioactive steroid sapogenin that is synthesized by a range of plant species (Jesus et al., 2016) and has been shown to bind in the orthosteric pocket of the chemo-tactile receptor from the striped pyjama squid (Sepioloidea lineolata) (van Giesen et al., 2020). As diosgenin is readily accessible, it has been extensively researched and acts at structurally related receptors where it was used as a substitute for ZINC36126889 and metagenin in functional studies. Bemcentinib, proscillaridin, and adapalene were also selected to examine functionally through electrophysiology and have predicted binding energies ≤ –11 kcal/mol. Based on their structural similarity to diosgenin, we further added the following analogs to our selection of compounds for testing in the electrophysiology assay: CHAPS, CHAPSO, and cholesteryl hemisuccinate (CHEMS). Finally, we also performed docking simulations with the neurotransmitters acetylcholine, GABA, and glycine, which were previously tested in a functional screen (Wijckmans et al., 2016). To determine the agonistic activity of hit compounds identified from virtual screening, all compounds were applied to oocytes injected with Alpo4 R" @default.
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• W4384074620 title "Decision letter: Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor" @default.
• W4384074620 doi "https://doi.org/10.7554/elife.86029.sa1" @default.
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