Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor
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eLife assessment
This study presents valuable structures of a pentameric ligand-gated ion channel from a thermophilic worm that is a homologue of the well-known mammalian nicotinic receptors. Although the function of the worm receptor is unknown, the authors convincingly identify interesting features for this class of receptors including a steroid detergent that is bound in the canonical neurotransmitter site and that induces conformational changes of the extracellular domains. These observations will be of broad interest to the ligand-gated ion channel community, although it is difficult at this moment to relate these observations to channel function as the channel's activating ligand remains unknown.
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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.
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Author Response
Reviewer #2 (Public Review):
In the discussion, the authors suggest that the binding of CHAPS could be an inspiration to develop compounds, targeting, for instance, mammalian receptors, that would bind to both the orthosteric site and a potential groove underneath loop C (where the sterol moiety of CHAPS binds in Alpo4). A figure (SI4) shows a few homologues in surface representation, giving an idea of whether this groove is generally present in the family.
Seeing this figure, I wondered if it would be relevant to compare several conformations of one or a few chosen homologues. Given that gating always impacts the quaternary assembly, is this groove more pronounced in say the inhibited state of a given homologue than in its agonist-bound state?
The width of the groove in 7 does change as the channel transition …
Author Response
Reviewer #2 (Public Review):
In the discussion, the authors suggest that the binding of CHAPS could be an inspiration to develop compounds, targeting, for instance, mammalian receptors, that would bind to both the orthosteric site and a potential groove underneath loop C (where the sterol moiety of CHAPS binds in Alpo4). A figure (SI4) shows a few homologues in surface representation, giving an idea of whether this groove is generally present in the family.
Seeing this figure, I wondered if it would be relevant to compare several conformations of one or a few chosen homologues. Given that gating always impacts the quaternary assembly, is this groove more pronounced in say the inhibited state of a given homologue than in its agonist-bound state?
The width of the groove in 7 does change as the channel transition from apo to open state. This is now demonstrated with an additional Figure 3 – figure supplement 1b and the discussion was adjusted accordingly p 18, line 379:
“The sterol group connected by a linker binds in between subunits and induces conformational changes which also change the width of the groove in Alpo4 (Figure 3f, g), therefore it likely plays an active role in the observed quaternary twist. The changes in the groove shape are not specific to Alpo4 but are also observed for example in nicotinic 7 receptor (Figure 3 – supplement 1b) suggesting that the groove can be targeted for allosteric modulation of the channel. ”
A related thought was that some of the protein binders affecting pLGIC function (toxins, VHH) contact two subunits and wrap around/below loop C. Do these have binding sites that overlap with the groove?
We inspected the structures of pLGICs homologs with bound -bungarotoxin (6UWZ, 4HQP, 7Z14, and 7KOO) and 2 with bound VHHs (6SSI and 6HJY). The toxins were bound in similar conformations but not the VHHs. The examples of the complexes are now shown as Supplementary Figure 13a (see above). In the case of ELIC, the nanobody Nb72 was bound on top of the sterol-binding cavity, but it did not interact with the interior of the cavity. This is now explained on p 17 from line 374:
“When binding sites of larger know binders, including VHH47,48 and -bungarotoxin10,49 were examined (Figure 3 – supplement 1a) a nanobody bound to ELIC in the site covering the sterol-binding groove was identified, however, its interactions with ELIC did not overlap significantly with the interior of the sterol-binding groove. This suggests that the latter is a novel target location for binders.”
Very interestingly, the binding of CHAPS stabilizes a conformation that differs from the apo one. It includes a twist of the ECDs but does not lead to a significant opening of the M2 bundle. The authors note that the direction of the twist is reversed to that often associated with the binding of ligands in homologues. This reversion is quite a feature, which deserves to be shown in a supplementary movie (e.g overlay of the Alpo apo>CHAPs transition with the nico>apo transition of a7).
We have re-examined the rotation and compared it to the conformational changes in nACh 7 and 5-HT3 receptors. Upon closer examination, it became clear that relative rotation of the ECD and the TMD provides a very simplistic view of the quaternary conformational changes which are more complex 3D quaternary changes than a simple relative domain rotation. Careful alignment of the structures to the extracellular side of the trans-membrane pore showed that in both channels resting-> open state transition is associated with clockwise rotation, but resting-> desensitized state transition in 5-HT3 involves a counterclockwise rotation. Thus, 1) the direction of rotation is not a ‘universal’ feature of pLGICs and 2) the clockwise rotation is the direction of channel activation for α7 nACh receptor and 5-HT3 and shares similarities with rearrangements observed in Alpo4. However, the relative movement of the ECDs is different between Alpo upon CHAPS binding and α7 nACh and 5-HT3 receptor upon activation. To demonstrate this, we added Video 2 which shows quaternary changes for all 3 channels and the text has been modified as follows on page 11 line 208:
“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.”
We are grateful to the reviewer for drawing our attention to this point, which permitted us to correct initially inaccurate statements.
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eLife assessment
This study presents valuable structures of a pentameric ligand-gated ion channel from a thermophilic worm that is a homologue of the well-known mammalian nicotinic receptors. Although the function of the worm receptor is unknown, the authors convincingly identify interesting features for this class of receptors including a steroid detergent that is bound in the canonical neurotransmitter site and that induces conformational changes of the extracellular domains. These observations will be of broad interest to the ligand-gated ion channel community, although it is difficult at this moment to relate these observations to channel function as the channel's activating ligand remains unknown.
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Reviewer #1 (Public Review):
The authors solved cryoEM structural maps for the pLGIC homolog Alpo4 from an extreme thermophile worm in apo and CHAPS-bound conditions. The data appear to be of good quality and in addition to a first structural model of Alpo4 also provide several interesting observations. Notably, the detergent CHAPS was observed to occupy the orthosteric binding pocket where it induces a quaternary twist of extracellular relative to transmembrane domains opposite to that observed for activation in canonical pLGICs. Given recent advances in lipid modulation of pLGICs, this structural model of how a detergent can bind to the orthosteric site will be of interest. Additionally, a ring of 16' methionines forms a potential alternate pore gate to the 9' leucine ring in canonical pLGICs. Unfortunately, testing these hypotheses …
Reviewer #1 (Public Review):
The authors solved cryoEM structural maps for the pLGIC homolog Alpo4 from an extreme thermophile worm in apo and CHAPS-bound conditions. The data appear to be of good quality and in addition to a first structural model of Alpo4 also provide several interesting observations. Notably, the detergent CHAPS was observed to occupy the orthosteric binding pocket where it induces a quaternary twist of extracellular relative to transmembrane domains opposite to that observed for activation in canonical pLGICs. Given recent advances in lipid modulation of pLGICs, this structural model of how a detergent can bind to the orthosteric site will be of interest. Additionally, a ring of 16' methionines forms a potential alternate pore gate to the 9' leucine ring in canonical pLGICs. Unfortunately, testing these hypotheses such as the alternate pore gate remains difficult due to the fact that the activating ligand for Alpo4 remains unknown. This also makes it hard to relate the observed changes upon CHAPS binding to the Alpo4 function. Nonetheless, the structures will aid in hypotheses for functional mechanisms of Alpo4 and pLGICs.
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Reviewer #2 (Public Review):
De Gieter et al.'s structural report follows a previous screening effort, which identified pLGIC from Alvinella pompejana as suitable for structural studies.
In the present manuscript, the authors report several structures of one homopentamer named Alpo4. The manuscript is organized around a thoughtful, convincing, description of the common points shared by Alpo4 with the mammalian homologues of known structures, and of its distinctive features. The most striking differences are 1. the unexpected presence of a CHAPS detergent molecule bound to the orthosteric site; 2. the unique rotamer switch of a conserved tryptophan in the apo binding pocket, creating what the authors call a 'self-liganded' state; 3. a tightly closed hydrophobic gate with a ring of methionine residues within the M2 helices. 4. A reversed …Reviewer #2 (Public Review):
De Gieter et al.'s structural report follows a previous screening effort, which identified pLGIC from Alvinella pompejana as suitable for structural studies.
In the present manuscript, the authors report several structures of one homopentamer named Alpo4. The manuscript is organized around a thoughtful, convincing, description of the common points shared by Alpo4 with the mammalian homologues of known structures, and of its distinctive features. The most striking differences are 1. the unexpected presence of a CHAPS detergent molecule bound to the orthosteric site; 2. the unique rotamer switch of a conserved tryptophan in the apo binding pocket, creating what the authors call a 'self-liganded' state; 3. a tightly closed hydrophobic gate with a ring of methionine residues within the M2 helices. 4. A reversed ECD twist associated with the binding of CHAPSThe principal strength of the manuscript is to extend the structural knowledge of the pLGIC family beyond the mammalian receptors to invertebrates, for which structural information has remained scarce. In particular, the binding of CHAPS to an 'extended' binding site is shown. That site does not only comprise the place where neurotransmitter usually binds but is prolonged by a hydrophobic patch underneath loop C and in contact with loop F/beta 8.
In the discussion, the authors suggest that the binding of CHAPS could be an inspiration to develop compounds, targeting for instance mammalian receptors, that would bind to both the orthosteric site and a potential groove underneath loop C (where the sterol moiety of CHAPS binds in Alpo4). A figure (SI4) shows a few homologues in surface representation, giving an idea of whether this groove is generally present in the family. Seeing this figure, I wondered if it would be relevant to compare several conformations of one or a few chosen homologues. Given that gating always impacts the quaternary assembly, is this groove more pronounced in say the inhibited state of a given homologue than in its agonist-bound state?
A related thought was that some of the protein binders affecting pLGIC function (toxins, VHH) contact two subunits and wrap around/below loop C. Do these have binding sites that overlap with the groove?Very interestingly, the binding of CHAPS stabilizes a conformation that differs from the apo one. It includes a twist of the ECDs but does not lead to a significant opening of the M2 bundle. The authors note that the direction of the twist is reversed to that often associated with the binding of ligands in homologues. This reversion is quite a feature, which deserves to be shown in a supplementary movie (e.g overlay of the Alpo apo>CHAPs transition with the nico>apo transition of a7). My mental framework was that in this family 1. inhibitors do not trigger much of a quaternary conformational change 2. agonists trigger changes always in the same direction (even if the amplitude and exact rotation vary from receptor to receptor). So it's interesting to see a compound (of unknown functional effect) triggering a reversed change.
The principal weakness of the manuscript lies in the absence of a known agonist for Alpo4, The authors do a good job at explaining what they tried and why (and they did perform quite an array of unsuccessful functional experiments), yet it remains frustrating to be unable to link the observed structures to some function.
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Reviewer #3 (Public Review):
Pentameric ligand-gated ion channels are a class of neurotransmitter receptors playing a key role in cellular communication. Besides their presence in mammalians, a multitude of receptors is found in lower organisms such as bacteria and invertebrates. They display a large diversity of molecular architectures and functions, as exemplified by atypical bacterial channels GLIC, ELIC, STELIC, or DeCLIC that have been characterized at the structural and functional levels. The study of unorthodox receptors, while challenging, is thus fascinating and is expected to give insights into the evolution, as well as the functional and structural divergence occurring in the superfamily.
In this work, authors solve the structure of the orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Alpo4 is …
Reviewer #3 (Public Review):
Pentameric ligand-gated ion channels are a class of neurotransmitter receptors playing a key role in cellular communication. Besides their presence in mammalians, a multitude of receptors is found in lower organisms such as bacteria and invertebrates. They display a large diversity of molecular architectures and functions, as exemplified by atypical bacterial channels GLIC, ELIC, STELIC, or DeCLIC that have been characterized at the structural and functional levels. The study of unorthodox receptors, while challenging, is thus fascinating and is expected to give insights into the evolution, as well as the functional and structural divergence occurring in the superfamily.
In this work, authors solve the structure of the orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Alpo4 is solved in two conformations, Apo and CHAPS-bound, both displaying a closed channel. The structures show several unusual features, in particular in the orthosteric site where, in the Apo, the tryptophan residues at the heart of the site lie in a place usually occupied by the neurotransmitter resembling a "self-liganded" conformation. In addition, the channel is bordered by unusual rings of hydrophobic residues in its upper part, and the protein shows substantial reorganization upon CHAPS binding. Alpo4 was previously investigated by electrophysiology but no agonist was found. Based on the structures, a number of gain-of-function mutants and chimeric constructs have been tested, but unfortunately, none are allowed to observe a ligand-gated ion channel function.
Overall, the paper is written in a very clear and fair manner, presenting the structural architecture and conformational reorganizations but also the limitation of the work concerning the lack of functional identification.
The paper constitutes a substantial amount of work (six cryo-EM structures in total). While it failed to identify an agonist and capture an open-channel conformation, the structure of a member of the family from an extreme thermophile species is novel and interesting for our fundamental knowledge of this important family of receptors.
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