Emergence of specific binding and catalysis from a designed generalist binding protein.

Binding and catalysis play central roles in living systems. While natural proteins have finely tuned affinities for their primary ligands, they also bind weakly and promiscuously to other molecules, which serve as starting points for the incremental evolution of different specificities. Thus, modern proteins have emerged from the joint exploration of sequence and structural space. Interactions between natural proteins and small molecules can be systematically profiled by crystallographic fragment screening in defined geometries, yet this approach has not been applied to highly designable de novo proteins. Here we apply this method to explore the binding specificity of a de novo small-molecule-binding protein, apixaban-binding helical bundle. As in nature, we found that it formed weak complexes, which were excellent starting points for the design of entirely distinct functions, including a turn-on fluorophore binder and a highly efficient Kemp eliminase with a catalytic efficiency of 3,200,000 M-1 s-1, approaching the diffusion limit. This work illustrates how simultaneous consideration of sequence and chemical structure diversity can guide the emergence of different functions in designed proteins.