Abstract
<jats:p>Many biological proteins function by changing shape upon small-molecule binding. Here, we present a general strategy for designing de novo proteins that undergo small-molecule-induced conformational change. Our approach converts a preorganized small-molecule binding protein into a ligand-responsive shape-changer by adding a mobile lid domain that closes behind the ligand upon binding. Using this strategy, we converted an exatecan-binding protein into a drug-induced conformational switch. The lidded proteins showed considerably stronger binding affinity in the sub-nanomolar regime, 100-fold greater specificity to exatecan over a similar molecule, and ligand residence times up to several months, with tunable binding kinetics. We turned one design into a genetically encodable fluorescent biosensor of the drug, enabling potential clinical applications. Our results open the door to programming complex molecular function using vast chemical space.</jats:p>