Biochemistry and Pharmacology - Research Publications
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ItemINPHARMA-Based Determination of Ligand Binding Modes at α1-Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity(WILEY-V C H VERLAG GMBH, 2020-09-10)The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α1A -AR and α1B -AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α1A -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α1A -AR and α1B -AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α1A -AR, thus demonstrating an approach for targeting subtype selectivity in ARs.
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ItemProbing the correlation between ligand efficacy and conformational diversity at the ?(1A)-adrenoreceptor reveals allosteric coupling of its microswitches(American Society for Biochemistry and Molecular Biology, 2020-05-22)G protein–coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.