Biology subjects

daCosta, C. J. B.

Publications and source records attributed to daCosta, C. J. B..

5 recordsLinked to original sources

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↗

bioRxiv2024

Ancestral sequence reconstruction of Mic60 reveals a residue signature supporting respiration in yeast

In eukaryotes, cellular respiration takes place in the cristae of mitochondria. The mitochondrial inner membrane protein Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS), is crucial for the organization and stabilization of crista junctions and its associated functions. While the C-terminal Mitofilin domain of Mic60 is necessary for cellular respiration, the sequence determinants for this function have remained unclear. Here, we used ancestral sequence reconstruction to generate Mitofilin ancestors up to and including the last opisthokont common ancestor (LOCA). We found that yeast-lineage derived Mitofilin ancestors as far back as the LOCA rescue respiration. By comparing Mitofilin ancestors, we identified four residues sufficient to explain the respiratory difference between yeast- and animal-derived Mitofilin ancestors. Our results provide a foundation for investigating the conservation of Mic60-mediated cristae junction interactions.

evolutionary biology↗

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↗