Determinants of ligand subtype-selectivity at α1A-adrenoceptor revealed using Saturation Transfer Difference (STD) NMR

Kelvin J Yong, Tasneem M Vaid, Patrick J Shilling, Feng-Jie Wu, Lisa M Williams, Mattia Deluigi, Andreas Plückthun, Ross AD Bathgate, Paul R. Gooley, Daniel J Scott

Index: 10.1021/acschembio.8b00191

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Abstract

α1A- and α1B-adrenoceptors (α1A-AR and α1B-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene super-family is made up of numerous sub-families that, like α1A-AR and α1B-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents however, makes the application of NMR to study ligand binding challenging. Here, stabilized α1A-AR and α1B-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α1A-AR over α1B-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.