Rapid Assessment of Chemical Complementarity of Ligands for Protein Design

Computational protein design is delivering de novo structures rapidly and reliably. This opens new frontiers for functional design, for instance, for binding small molecules tightly and specifically. However, achieving predictable and tuneable binding is challenging. Here we introduce a rapid physics-based method to generate isosteric and chemically complementary binding pockets for small-molecule targets in de novo proteins. We test this by constructing and characterizing binding proteins for several synthetic and natural chromophores. By evaluating only single-digit numbers of designs, the pipeline delivers stable proteins with pre-organised binding sites confirmed by X-ray crystallography, which bind the targets with micromolar affinities or better. To illustrate the scope and applications of this approach, we incorporate selective and coupled chromophore-binding sites in a two-domain de novo protein enabling controlled energy transfer between the two sites, and we develop a small de novo binding protein that can be used in live mammalian cells to visualise sub-cellular structures.