Cellular signal transduction is predominantly based on protein interactions and their posttranslational modifications, which enable a fast response to input signals. Due to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, this has a substantially slower response requiring transcription and translation. Here, we present a de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled-coils (CC) implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains of tunable affinity for competitive displacement after proteolytic cleavage. This enabled implementation of Boolean logic functions and signaling cascades in mammalian cells. Designed split proteasecleavable orthogonal CC-based logic (SPOC logic) circuits enable response to chemical or biological signals within minutes rather than hours, useful for diverse medical and nonmedical applications.
Protein interactions guide the majority of cellular processes. Orthogonal hetero-specific proteinprotein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR/dCas9-based transcriptional activators and to amplify the response of light-and small-molecule inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.
The coiled-coil dimer is a widespread protein structural motif and, due to its designability, represents an attractive building block for assembling modular nanostructures. The specificity of coiled-coil dimer pairing is mainly based on hydrophobic and electrostatic interactions between residues at positions a, d, e, and g of the heptad repeat. Binding affinity, on the other hand, can also be affected by surface residues that face away from the dimerization interface. Here we show how design of the local helical propensity of interacting peptides can be used to tune the stabilities of coiled-coil dimers over a wide range. By designing intramolecular charge pairs, regions of high local helical propensity can be engineered to form trigger sequences, and dimer stability is adjusted without changing the peptide length or any of the directly interacting residues. This general principle is demonstrated by a change in thermal stability by more than 30 °C as a result of only two mutations outside the binding interface. The same approach was successfully used to modulate the stabilities in an orthogonal set of coiled-coils without affecting their binding preferences. The stability effects of local helical propensity and peptide charge are well described by a simple linear model, which should help improve current coiled-coil stability prediction algorithms. Our findings enable tuning the stabilities of coiled-coil-based building modules match a diverse range of applications in synthetic biology and nanomaterials.
Coiled-coil (CC) dimers are versatile, customizable building modules for the design of diverse protein architectures unknown in nature. Incorporation of dynamic self-assembly, regulated by a selected chemical signal, represents an important challenge in the construction of functional polypeptide nanostructures. Here, we engineered metal binding sites to render an orthogonal set of CC heterodimers Zn(II)-responsive as a generally applicable principle. The designed peptides assemble into CC heterodimers only in the presence of Zn(II) ions, reversibly dissociate by metal ion sequestration, and additionally act as pH switches, with low pH triggering disassembly. The developed Zn(II)-responsive CC set is used to construct programmable folding of CC-based nanostructures, from protein triangles to a two-chain bipyramidal protein cage that closes and opens depending on the metal ion. This demonstrates that dynamic self-assembly can be designed into CC-based protein cages by incorporation of metal ion–responsive CC building modules that act as conformational switches and that could also be used in other contexts.
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