GPCR engineering yields high-resolution structural insights into β₂-adrenergic receptor function

The β₂-adrenergic receptor (β₂AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the β₂AR and to facilitate its crystallization, we engineered a β₂AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("β₂AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of β₂AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.