De Novo Design of Bioactive Protein Switches

Allosteric regulation of protein function is widespread in biology, but challenging for de novo protein design as it requires explicit design of multiple states with comparable free energies. We explore the possibility of de novo designing switchable protein systems through modulation of competing inter and intra-molecular interactions. We design a static, five-helix “Cage” with a single interface that can interact either intra-molecularly with a terminal “Latch” helix or inter-molecularly with a peptide “Key”. Encoded on the Latch are functional motifs for binding, degradation, or nuclear export that function only when the Key displaces the Latch from the Cage. We describe orthogonal Cage-Key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by Key. The design of switchable protein function controlled by induced conformational change is a milestone for de novo protein design and opens up new avenues for synthetic biology and cell engineering.