In Vitro Evolution of the Adenosine A_2A Receptor Based on an Antagonist Binding Using a Ribosome Display

In vitro 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 in vitro. This study presents the first successful in vitro evolution of human adenosine A_2A receptor (A_2AR), 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 A_2AR 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 A_2AR-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 A_2AR 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 A_2AR signaling by ZM241385. This study demonstrates a powerful strategy for engineering GPCRs entirely in vitro with tailored ligand-binding properties, even from a library with small diversity, and shows that the evolved mutants exhibit improved functionality in mammalian cells.