Natarajan, Kathiresan

Background and Purpose α4β2 nicotinic acetylcholine (nACh) receptors assemble in two stoichiometric forms, one of which is potentiated by calcium. The sites of calcium binding that underpin potentiation are not known. Experimental Approach To identify calcium binding sites, we applied cryo‐electron microscopy (cryo‐EM) and molecular dynamics (MD) simulations to each stoichiometric form of the α4β2 nACh receptor in the presence of calcium ions. To test whether the identified calcium sites are linked to potentiation, we generated mutants of anionic residues at the sites, expressed wild type and mutant receptors in clonal mammalian fibroblasts, and recorded ACh‐elicited single‐channel currents with or without calcium. Key Results Both cryo‐EM and MD simulations show calcium bound to a site between the extracellular and transmembrane domains of each α4 subunit (ECD‐TMD site). Substituting alanine for anionic residues at the ECD‐TMD site abolishes stoichiometry‐selective calcium potentiation, as monitored by single‐channel patch clamp electrophysiology. Additionally, MD simulation reveals calcium association at subunit interfaces within the extracellular domain. Substituting alanine for anionic residues at the ECD sites reduces or abolishes stoichiometry‐selective calcium potentiation. Conclusions and Implications Stoichiometry‐selective calcium potentiation of the α4β2 nACh receptor is achieved by calcium association with topographically distinct sites framed by anionic residues within the α4 subunit and between the α4 and β2 subunits. Stoichiometry‐selective calcium potentiation could result from the greater number of calcium sites in the stoichiometric form with three rather than two α4 subunits. The results are relevant to modulation of signalling via α4β2 nACh receptors in physiological and pathophysiological conditions.

AbstractThe mainstream approach to antiviral drugs against COVID‐19 is to focus on key stages of the SARS‐CoV‐2 life cycle. The vast majority of candidates under investigation are repurposed from agents of other indications. Understanding protein–inhibitor interactions at the molecular scale will provide crucial insights for drug discovery to stop this pandemic. In this article, we summarize and analyze the most recent structural data on several viral targets in the presence of promising inhibitors for COVID‐19 in the context of the perspective of modes of action (MOA) to unravel insightful mechanistic features with atomistic resolution. The targets include spike glycoprotein and various host proteases mediating the entry of the virus into the cells, viral chymotrypsin‐ and papain‐like proteases, and RNA‐dependent RNA polymerase. The main purpose of this review is to present detailed MOA analysis to inspire fresh ideas for both de novo drug design and optimization of known scaffolds to combat COVID‐19.

AbstractThe mainstream approach to antiviral drugs against COVID‐19 is to focus on key stages of the SARS‐CoV‐2 life cycle. The vast majority of candidates under investigation are repurposed from agents of other indications. Understanding protein–inhibitor interactions at the molecular scale will provide crucial insights for drug discovery to stop this pandemic. In this article, we summarize and analyze the most recent structural data on several viral targets in the presence of promising inhibitors for COVID‐19 in the context of the perspective of modes of action (MOA) to unravel insightful mechanistic features with atomistic resolution. The targets include spike glycoprotein and various host proteases mediating the entry of the virus into the cells, viral chymotrypsin‐ and papain‐like proteases, and RNA‐dependent RNA polymerase. The main purpose of this review is to present detailed MOA analysis to inspire fresh ideas for both de novo drug design and optimization of known scaffolds to combat COVID‐19.

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.