From synthetic coiled coils to functional proteins: Automated design of a receptor for the calmodulin-binding domain of calcineurin

A series of synthetic receptors capable of binding to the calmodulin-binding domain of calcineurin (CN_393-414) was designed, synthesized and characterized. The design was accomplished by docking CN_393-414 against a two-helix receptor, using an idealized three-stranded coiled coil as a starting geometry. The sequence of the receptor was chosen using a side-chain re-packing program, which employed a genetic algorithm to select potential binders from a total of 7.5 x 10⁶ possible sequences. A total of 25 receptors were prepared, representing 13 sequences predicted by the algorithm as well as 12 related sequences that were not predicted. The receptors were characterized by CD spectroscopy, analytical ultracentrifugation, and binding assays. The receptors predicted by the algorithm bound CN_393-414 with apparent dissociation constants ranging from 0.2 μM to > 50 μM. Many of the receptors that were not predicted by the algorithm also bound to CN_393-414. Methods to circumvent this problem and to improve the automated design of functional proteins are discussed.