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

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• * In vitro evolution is powerful for protein modification but challenging for membrane proteins.
• * Membrane protein engineering requires an *in vitro* membrane environment.

Purpose of the Study:

• * To achieve the first successful *in vitro* evolution of the human adenosine A2A receptor (A2AAR).
• * To engineer A2AAR for tailored ligand-binding properties and improved functionality.

Main Methods:

• * Combined cell-free protein synthesis (CFPS), nanodisc technology for membrane mimicry, and ribosome display.
• * Constructed a saturation mutagenesis library targeting A2AAR residues L167 and L267.
• * Performed three rounds of affinity selection using A2AAR-selective antagonist ZM241385.

Main Results:

• * Enriched L167N/L267A and L167N/L267S A2AAR mutants.
• * Mutants showed >10-fold increased sensitivity to ZM241385 in cell-based assays.
• * Retained responsiveness to endogenous adenosine and demonstrated improved complex stability via molecular dynamics.

Conclusions:

• * Developed a robust *in vitro* strategy for engineering G protein-coupled receptors (GPCRs).
• * Evolved A2AAR mutants exhibit enhanced ligand-binding properties and functionality in mammalian cells.
• * Demonstrated cell-type-specific inhibition of A2AAR signaling using an engineered mutant.