Biology subjects

Tessier, C. J. G.

Publications and source records attributed to Tessier, C. J. G..

6 recordsLinked to original sources

bioRxiv2025

An extended structure of the intracellular domain of the Torpedo nicotinic acetylcholine receptor and its proposed interactions with rapsyn

To gain insight into the interactions between rapsyn and the nAChR that induce clustering at the post-synaptic membrane, we refined a cryo-EM dataset using an intracellular domain focused strategy to obtain a 3.0 [A] map with the most extensive density yet for the intracellular domain of the Torpedo nAChR. The improved map allowed us to extend the structure beyond the MX -helix and prior to the MA -helix of the intracellular domain. The new structure defines a sharp N-terminal boundary of each MA -helix to place agrin-dependent phosphorylated tyrosines unambiguously within the flexible regions of the MX-MA loops. Two distinct conformations of the {delta} M4 -helix were also resolved, indicating that M4 conformational heterogeneity reflects intrinsic flexibility rather than a change in gating state. The new structural constraints defined for the MX-MA loop were then used to evaluate AlphaFold3-predicted full-length models of the nAChR, rapsyn, and various rapsyn-nAChR complexes, identifying a consistent, asymmetric 3:1 binding architecture where each rapsyn is always sandwiched between the MX-MA loops from two subunits and where each phospho-tyrosine is lodged in a cationic pocket formed by conserved residues implicated in congenital myasthenic syndromes. The defined architecture fits published cryo-ET maps of Torpedo post-synaptic membranes and explains how both phosphorylated tyrosines and myasthenic syndrome-causing rapsyn mutations modulate receptor clustering.

neuroscience↗

bioRxiv2025

Heteropentameric architecture predisposes functional inequivalence of acetylcholine receptor agonist sites

Muscle-type acetylcholine receptors are heteropen-tameric ion channels composed of four different but evolutionarily related subunits. These subunits assemble with a precise stoichiometry and arrangement, such that two distinct agonist-binding sites are formed at interfaces between a principal -subunit and a complementary {delta} or {varepsilon}/{gamma}-subunit. Chemical differences between the two complementary subunits are presumed to confer functional inequivalence to the two agonist sites. This interpretation, however, overlooks the asymmetric architecture of the acetylcholine receptor, which places each subunit, and therefore each agonist site, at unique positions within the heteropentamer. The extent to which functional inequivalence of the agonist sites stems from this structural asymmetry, as opposed to chemical differences, remains unexplored. Here, we reconstruct an ancestral subunit capable of substituting for both complementary subunits, thereby engineering hybrid ancestral/human acetylcholine receptors with chemically identical agonist sites. Despite being chemically identical, these agonist sites remain functionally inequivalent. We show that this functional inequivalence stems from distinct intersubunit interactions dictated by the receptors heteropentameric architecture, which also underlie the subunit-dependent effects of a human disease-causing mutation. Thus, structural asymmetry emerges as a fundamental determinant of receptor function, capable of imposing functional inequivalence even in chemically identical sites, with implications for both the evolution of heteromeric protein complexes and the molecular basis of disease.

biophysics↗

bioRxiv2024

Asynchronous subunit transitions precede acetylcholine receptor activation

Rapid communication at synapses is facilitated by postsynaptic receptors, which convert a chemical signal into an electrical response. In the case of ligand-gated ion channels, agonist binding triggers rapid transition through a series of intermediate states leading to a transient open-pore conformation. These transitions are usually framed in terms of a mechanism where agonist binding and channel activation are separate events. Here, we collect cryo-EM images over a range of agonist concentrations to define structures of the muscle-type nicotinic acetylcholine receptor in unliganded, mono-liganded, and di-liganded states. We show that agonist binding to a single agonist site stabilizes an intermediate state where an entire principal agonist-binding subunit has transitioned to an active-like conformation, while the other unoccupied principal subunit remains inactive, albeit poised for activation. Binding of agonist to the second agonist site fully activates the remaining subunits leading to hydration of the ion pore. Uniting this cryo-EM derived intermediate structure with single-channel recordings leads to a model where individual acetylcholine receptor subunits asynchronously undergo conformational transitions, and thus a sequential activation mechanism that has implications for the entire superfamily of pentameric ligand-gated ion channels.

neuroscience↗

bioRxiv2023

pUdOs: concise plasmids for bacterial and mammalian cells

The pUdOs are 28 plasmids of small size combining four different origins of replication and seven selection markers, which together afford flexible use in Escherichia coli and several related gram- negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators, and contains type IIP or IIS restriction sites for conventional or Golden-gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the users discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors affording distinct levels of expression and transfection efficiencies comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

synthetic biology↗

bioRxiv2022

Derepression masquerades as activation in a pentameric ligand-gated ion channel

Agonists are ligands that bind to receptors and activate them. In the case of ligand-gated ion channels, such as the muscle-type nicotinic acetylcholine receptor, mechanisms of agonist activation have been studied for decades. Taking advantage of a reconstructed ancestral muscle-type {beta}-subunit that forms spontaneously activating homopentamers, here we show that incorporation of human muscle-type -subunits represses spontaneous activity, and furthermore that the presence of agonist relieves this -subunit-dependent repression. Our results demonstrate that rather than provoking channel activation/opening, agonists may instead inhibit the inhibition of intrinsic spontaneous activity. Thus, agonist activation may be the apparent manifestation of agonist-induced derepression. These results provide insight into intermediate states that precede channel opening and have implications for the interpretation of agonism in ligand-gated ion channels.

biophysics↗

bioRxiv2022

Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two -subunits, and one each of the {beta}-, {delta}-, and {varepsilon}-subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we show that a reconstructed ancestral muscle-type {beta}-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings demonstrate that signature features of muscle-type acetylcholine receptor function are independent of agonist, and do not necessitate the complex heteropentameric architecture of the modern-day receptor.

biophysics↗