In Vitro Evolution of the Adenosine A(2A) Receptor Based on an Antagonist Binding Using a Ribosome Display.

evolution is one of the powerful methods for altering the characteristics of a protein of interest; however, its application to membrane proteins remains challenging due to the requirement of a membrane environment . This study presents the first successful evolution of human adenosine A receptor (AR), one of the G protein-coupled receptors (GPCRs), achieved through combining cell-free protein synthesis (CFPS), a nanodisc for membrane mimicry, and a ribosome display. A saturation mutagenesis library of AR targeting the L167 and L267 residues, a library with a diversity of only 400, was constructed without sequence bias. Three rounds of affinity selection against AR-selective antagonist ZM241385 enriched the L167N/L267A and L167N/L267S mutants. These mutants exhibited greater than 10-fold improved sensitivity to ZM241385 in mammalian cell-based assays while retaining responsiveness to the endogenous ligand adenosine. Molecular dynamics simulations revealed new interactions between ZM241385 and AR mutants that are likely to contribute to stabilizing the conformation of the complexes. Finally, as an example of an application, the obtained mutant was used for cell-type-specific inhibition of AR signaling by ZM241385. This study demonstrates a powerful strategy for engineering GPCRs entirely with tailored ligand-binding properties, even from a library with small diversity, and shows that the evolved mutants exhibit improved functionality in mammalian cells.