Biology subjects

Thomson, N. J.

Publications and source records attributed to Thomson, N. J..

5 recordsLinked to original sources

bioRxiv2026

Adenosine impairs T cell function via activation of purine salvage pathway and AMP-induced inhibition of pyrimidine nucleotide biosynthesis

Extracellular adenosine (ADO) is a well-established immune checkpoint mediator suppressing T cells via activation of A2A receptors, yet intracellular mechanisms of ADO action remain less understood. Here, we show that activated human T cells transport extracellular ADO through equilibrative nucleoside transporter 1 (ENT1) and metabolize it through ATP salvage pathway. The generated ADO metabolite AMP inhibits UMP synthase (UMPS), the rate-limiting enzyme of de novo pyrimidine biosynthesis, leading to diminished ATP production via conventional bioenergetic pathways, increased apoptosis, and impaired effector functions of T cells. Flow cytometric and single-cell transcriptomic analyses further revealed that ADO restrains the exit of naive T cells from quiescence. All effects of ADO were phenocopied by treating T cells with the inhibitors of UMPS and reversed after pharmacological blockage of ENT1 or uridine supplementation. Additional molecular docking, molecular dynamics simulations, and free-energy analyses indicate that AMP can occupy the orotidine 5'-monophosphate (OMP) decarboxylase catalytic site of UMPS with the same anchoring contacts as substrate OMP and product UMP, providing a structural rationale for direct AMP-mediated inhibition of pyrimidine biosynthesis. These findings demonstrate that immunosuppressive effects of ADO extend beyond canonical adenosinergic signaling and involve cellular ADO uptake and modulation of purine and pyrimidine metabolism.

immunology↗

bioRxiv2023

Mechanism of negative μ-opioid receptor modulation by sodium ions

The negative allosteric modulation of G-protein coupled receptors (GPCRs) by Na+ ions, known as the sodium effect, was first described in the 1970s for opioid receptors (ORs). Since then, it has been detected almost universally amongst class A GPCRs. High-resolution structures of class A GPCRs in the inactive state exhibit a Na+ ion bound to a conserved pocket near residue D2.50, whereas the active state structures of GPCRs are incompatible with Na+ binding. Correspondingly, Na+ ions diminish the affinity of receptor agonists, stabilize the receptors in the inactive state, and reduce basal signalling levels. Despite these observations, a detailed mechanistic explanation of how Na+ ions negatively modulate the receptor and inhibit activation has remained elusive. Here, we apply a mutual-information based analysis method to s-timescale all-atom molecular dynamics simulations of the -OR to decipher conformational changes within the protein matrix and protein-internal water molecules that are directly coupled to the binding of Na+. Our results reveal that Na+ binding is tightly coupled to a water wire that links the Na+ binding site with the agonist binding pocket. Furthermore, Na+ binding leads to rearrangements in polar protein networks that propagate conformational changes both to the agonist and the intracellular G-protein binding sites via conserved micro-switch motifs. Our findings provide a mechanistic link between the presence of the ion and altered agonist binding affinity, receptor deactivation and the depression of basal signalling.

biophysics↗

bioRxiv2023

The molecular mechanism of monovalent cation selectivity in the TRPM5 channel

A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting Transient Receptor Potential (TRP) superfamily of ion channels form a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca2+, TRPM4 and TRPM5 are unique due to their strong monovalent-selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent-selectivity by in silico electrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity rather than the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance ("hydrophobic funnel") enables monovalent, but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a co-operative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favourably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5.

biophysics↗

bioRxiv2022

A co-operative knock-on mechanism underpins Ca2+-selective cation permeation in TRPV channels

The selective exchange of ions across cellular membranes is a vital biological process. Ca2+-mediated signalling is implicated in a broad array of physiological processes in cells, whilst elevated intracellular concentrations of Ca2+ are cytotoxic. Due to the significance of this cation, strict Ca2+ concentration gradients are maintained across the plasma and organelle membranes. Therefore, Ca2+ signalling relies on permeation through selective ion channels that control the flux of Ca2+ ions. A key family of Ca2+-permeable membrane channels are the polymodal signal-detecting Transient Receptor Potential (TRP) ion channels. TRP channels are activated by a wide variety of cues including temperature, small molecules, transmembrane voltage and mechanical stimuli. Whilst most members of this family permeate a broad range of cations non-selectively, TRPV5 and TRPV6 are unique due to their strong Ca2+-selectivity. Here, we address the question of how some members of the TRPV subfamily show a high degree of Ca2+-selectivity whilst others conduct a wider spectrum of cations. We present results from all-atom molecular dynamics simulations of ion permeation through two Ca2+-selective and two non-selective TRPV channels. Using a new method to quantify permeation co-operativity based on mutual information, we show that Ca2+-selective TRPV channel permeation occurs by a three binding site knock-on mechanism, whereas a two binding site knock-on mechanism is observed in non-selective TRPV channels. Each of the ion binding sites involved displayed greater affinity for Ca2+ over Na+. As such, our results suggest that coupling to an extra binding site in the Ca2+-selective TRPV channels underpins their increased selectivity for Ca2+ over Na+ ions. Furthermore, analysis of all available TRPV channel structures shows that the selectivity filter entrance region is wider for the non-selective TRPV channels, slightly destabilising ion binding at this site, which is likely to underlie mechanistic decoupling.

biophysics↗

bioRxiv2020

Ion-water coupling controls class A GPCR signal transduction pathways

G-protein-coupled receptors (GPCRs) transmit signals across the cell membrane, forming the largest family of membrane proteins in humans. Most GPCRs activate through an evolutionarily conserved mechanism, which involves reorientation of helices and key residues, rearrangement of a hydrogen bonding network mediated by water molecules, and the expulsion of a sodium ion from a protonatable binding site. However, how these components interplay to engage the signal effector binding site remains elusive. Here, we applied information theory to molecular dynamics simulations of pharmaceutically important GPCRs to trace concerted conformational variations across the receptors. We discovered a conserved communication pathway that includes protein residues and cofactors and enables the exchange of information between the extracellular sodium binding site and the intracellular G-protein binding region, coupling the most highly conserved protonatable residues at long distance. Reorientation of internal water molecules was found to be essential for signal transmission along this pathway. By inhibiting protonation, sodium decoupled this connectivity, identifying the ion as a master switch that determines the receptors ability to move towards active conformations.

biophysics↗