A rational blueprint for the design of chemically-controlled protein switches

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.

Connexons Coupling to Gap Junction Channel: Potential Role for Extracellular Protein Stabilization Centers

“…Previously, we reported that known peptide inhibitors, mimicking a variety EL1 or EL2 sequences of Cx43 subtype, appear non-selective when binding to Cx43 versus Cx36 GJC subtypes [ 15 ]. In searching for potentially Cx subtype-specific inhibitors, we devised a strategy to design protein components and/or small molecules involved in the inhibition of protein-protein interactions [ 16 , 17 , 18 ]. Here we explore the prospective approach to inhibit connexon coupling by targeting distinguishable Cx subype-specific structural motifs within the GJ domain.…”

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

“…37,39,[85][86][87] Moreover, because our designed peptides and protein assemble efficiently in cells, such as E. coli, we anticipate applications to intervene in and to augment natural sub-cellular processes. 10,53,58,62,88,89 In short, we believe that our work adds fundamental understanding of the structural principles and sequence-to-structure relationships for coiled coils generally and 4-helix bundles specifically; and that our new designs provide platforms for future de novo design, and chemical and synthetic biology programs.…”

“…37, 39, 85–87 Moreover, because our designed peptides and protein assemble efficiently in cells, such as E. coli , we anticipate applications to intervene in and to augment natural sub-cellular processes. 10, 53, 58, 62, 88, 89…”

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

“…37,39,[85][86][87] Moreover, because our designed peptides and protein assemble efficiently in cells, such as E. coli, we anticipate applications to intervene in and to augment natural subcellular processes. 10,53,58,62,88,89 In short, we posit that our work adds fundamental understanding of the structural principles and sequence-to-structure relationships for coiled coils generally and 4-helix bundles specically; and that our new designs provide platforms for future de novo design, and chemical and synthetic biology programs.…”