Protein interactions guide most cellular processes. Orthogonal hetero-specific protein–protein 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 (called 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.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
Kim, K. H., Chandran, D. & Sauro, H. M. in Design and Analysis of Biomolecular Circuits (eds Koeppl, H., Gianluca Setti, G., di Bernardo M. & Densmore, D.) 117–138 (Springer, 2011).
Kuriyan, J. & Cowburn, D. Modular peptide recognition domains in eukaryotic signaling. Annu. Rev. Biophys. Biomol. Struct. 26, 259–288 (1997).
Burkhard, P., Stetefeld, J. & Strelkov, S. V. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 82–88 (2001).
Thompson, K. E., Bashor, C. J., Lim, W. A. & Keating, A. E. SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains. ACS Synth. Biol. 1, 118–129 (2012).
Fekonja, O., Benčina, M. & Jerala, R. Toll/Interleukin-1 receptor domain dimers as the platform for activation and enhanced inhibition of Toll-like receptor signaling. J. Biol. Chem. 287, 30993–31002 (2012).
Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).
Luan, H., Peabody, N. C., Vinson, C. R. & White, B. H. Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression. Neuron 52, 425 (2006).
Selgrade, D. F., Lohmueller, J. J., Lienert, F. & Silver, P. A. Protein scaffold-activated protein trans-splicing in mammalian cells. J. Am. Chem. Soc. 135, 7713–7719 (2013).
Tripet, B. et al. Engineering a de novo-designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins. Protein Eng. Des. Sel. 9, 1029–1042 (1996).
Yano, Y. et al. Coiled-coil tag–probe system for quick labeling of membrane receptors in living cells. ACS Chem. Biol. 3, 341–345 (2008).
Yano, Y. & Matsuzaki, K. Live-cell imaging of membrane proteins by a coiled-coil labeling method—principles and applications. Biochim. Biophys. Acta—Biomembr. 1861, 1011–1017 (2019).
Woolfson, D. N. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 70, 79–112 (2005).
Kaplan, J. B., Reinke, A. W. & Keating, A. E. Increasing the affinity of selective bZIP-binding peptides through surface residue redesign. Protein Sci. 23, 940–953 (2014).
Drobnak, I., Gradišar, H., Ljubetič, A., Merljak, E. & Jerala, R. Modulation of coiled-coil dimer stability through surface residues while preserving pairing specificity. J. Am. Chem. Soc. 139, 8229–8236 (2017).
Ljubetič, A. et al. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat. Biotechnol. 35, 1094–1101 (2017).
Chen, Z. et al. Programmable design of orthogonal protein heterodimers. Nature 565, 106–111 (2019).
Cho, J. H., Collins, J. J. & Wong, W. W. Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell 173, 1426–1438.e11 (2018).
Gradišar, H. & Jerala, R. De novo design of orthogonal peptide pairs forming parallel coiled-coil heterodimers. J. Pept. Sci. 17, 100–106 (2011).
Gradišar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat. Chem. Biol. 9, 362–366 (2013).
O’Shea, E. K., Lumb, K. J. & Kim, P. S. Peptide ‘Velcro’: design of a heterodimeric coiled coil. Curr. Biol. 3, 658–667 (1993).
Shekhawat, S. S., Porter, J. R., Sriprasad, A. & Ghosh, I. An autoinhibited coiled-coil design strategy for split-protein protease sensors. J. Am. Chem. Soc. 131, 15284–15290 (2009).
Thomas, F., Boyle, A. L., Burton, A. J. & Woolfson, D. N. A set of de novo designed parallel heterodimeric coiled coils with quantified dissociation constants in the micromolar to sub-nanomolar regime. J. Am. Chem. Soc. 135, 5161–5166 (2013).
Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).
Gao, Y. et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat. Methods 13, 1043–1049 (2016).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).
Mansouri, M., Strittmatter, T. & Fussenegger, M. Light-controlled mammalian cells and their therapeutic applications in synthetic biology. Adv. Sci. 6, 1800952 (2019).
Wu, C.-Y., Roybal, K. T., Puchner, E. M., Onuffer, J. & Lim, W. A. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science 350, aab4077 (2015).
Smole, A., Lainšček, D., Bezeljak, U., Horvat, S. & Jerala, R. A synthetic mammalian therapeutic gene circuit for sensing and suppressing inflammation. Mol. Ther. 25, 102–119 (2017).
Lonzarić, J., Lebar, T., Majerle, A., Manček-Keber, M. & Jerala, R. Locked and proteolysis-based transcription activator-like effector (TALE) regulation. Nucleic Acids Res. 44, 1471–1481 (2016).
Potapov, V., Kaplan, J. B., Keating, A. E., Howlett, G. & Schubert, D. Data-driven prediction and design of bZIP coiled-coil interactions. PLoS Comput. Biol. 11, e1004046 (2015).
Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).
Kis, Z., Pereira, H. S., Homma, T., Pedrigi, R. M. & Krams, R. Mammalian synthetic biology: emerging medical applications. J. R. Soc. Interface 12, 20141000 (2015).
Weber, W. & Fussenegger, M. Emerging biomedical applications of synthetic biology. Nat. Rev. Genet. 13, 21–35 (2012).
Kemmer, C. et al. Self-sufficient control of urate homeostasis in mice by a synthetic circuit. Nat. Biotechnol. 28, 355–360 (2010).
Wasilewska, A. et al. An update on abscisic acid signaling in plants and Mor. Mol. Plant. 1, 198–217 (2008).
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Breuza, L. et al. The UniProtKB guide to the human proteome. Database 2016, bav120 (2016).
UniProt Consortium, T. U. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 47, D506–D515 (2019).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).
Kwak, S. K. & Kim, J. H. Statistical data preparation: management of missing values and outliers. Korean J. Anesthesiol. 70, 407–411 (2017).
This research was supported by grants from the Slovenian Research Agency (nos. P4-0176, J1-9173, J3-7034 and N4-0080), ERC grant MaCChines to R.J, Horizon2020 CSA Bioroboost and ERANET project MediSurf. T.L. is partially supported by the UNESCO-L’OREAL national fellowship ‘For Women in Science’. We thank H. Gradišar for providing the sequences of CC peptides and for valuable advice.
The authors declare no competing interests.
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Lebar, T., Lainšček, D., Merljak, E. et al. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-019-0443-y