Abstract
Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.
Highlights
Small-molecule responsive protein switches are crucial components to control synthetic cellular activities
We described the design of chemicallydisruptable heterodimers (CDH)-1 composed of the BclXL:Lead Design 3 (LD3) complex which dissociates in the presence of the Bcl-XL -specific inhibitor A-1155463 (Drug-1)
We found that LD3 binds to Bcl[2] (Fig. 2a), a protein from the Bcl[2] family[25] closely related to BclXL, with a dissociation constant (Kd) of 0.8 nM as determined by surface plasmon resonance (SPR) (Fig. 2b)
Summary
Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches. For CIDs, Hill and colleagues used an in vitro evolution-based approach to engineer antibodies that engage with Bcl-XL only in the presence of a small-molecule drug[11], and showed that these switches were active in cellular applications. Foight and colleagues used libraries of computationally designed mutants of a previously reported de novo protein scaffold to interact with a viral protease only in its drugbound state[12] These were shown to regulate cellular activities in vivo, but the crystal structures of the designs evidenced substantial differences to the predicted binding modes. The precise design of key interaction residues to mediate small molecule interactions and control CIDs remain an extremely challenging computational design problem
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