Comparing the conformational diversity of α1A-Adrenoceptor in Micelles and Phospholipid Bilayer Models

Abstract

1A-adrenoceptor (1A-AR) is a class A G-protein coupled receptor (GPCR) that stimulates smooth muscle contraction in response to adrenaline and noradrenaline. GPCRs exist in a dynamic equilibrium between multiple conformational states. Ligand binding induces structural rearrangements via conserved microswitches, which are thought to shift the equilibrium and trigger signalling. For structural and biochemical studies, GPCRs must be solubilised from the membrane, typically using detergent micelles. However, detergents can disrupt native dynamics of membrane proteins, potentially confounding experimental results. To address this, phospholipid bilayer mimetics such as nanodiscs and saposin nanoparticles (SNPs) have been developed to provide a more native-like environment. Thermostabilised 1A-AR serves as a GPCR prototype and can be expressed and isotopically labelled for NMR purposes. To investigate how membrane mimetics influence the conformational diversity of 1A-AR, we compared 1H 13C3-HMQC NMR experiments of 13CH3-Met labelled 1A-AR incorporated into either DDM, LMNG, or SNPs, in presence of ligands with varying efficacies. Several methionine residues are positioned near key microswitches, including M2035.57, located closed to the G protein binding site. Its resonance has been proposed as a readout of receptor conformational state, shifting with ligand efficacy. Spectra of 13CH3-Met labelled 1A-AR in LMNG closely resembled those in DDM with some temperature-dependent dynamic variation. In contrast, incorporation into SNPs led to a complete loss of M2035.57 signal, consistent with an intermediate exchange rate. These findings demonstrate that the membrane environment can profoundly influence conformational dynamics in GPCR NMR studies. Our results highlight the need to carefully consider membrane environment when interpreting NMR data and underscore the value of benchmarking against biologically relevant controls.