Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Orthogonality of the designed NICP peptide set in HEK293T cells.
Fig. 2: CC-directed localization of multiple proteins.
Fig. 3: Tunability of the interaction strength within the NICP set toolbox.
Fig. 4: Highly potent designed CRISPR–CCC transcriptional activation platform.
Fig. 5: Enhancement of conditionally regulated transcription with CRISPR–CCC.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. 1.

    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).

  2. 2.

    Kuriyan, J. & Cowburn, D. Modular peptide recognition domains in eukaryotic signaling. Annu. Rev. Biophys. Biomol. Struct. 26, 259–288 (1997).

    CAS  Article  Google Scholar 

  3. 3.

    Burkhard, P., Stetefeld, J. & Strelkov, S. V. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 82–88 (2001).

    CAS  Article  Google Scholar 

  4. 4.

    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).

    CAS  Article  Google Scholar 

  5. 5.

    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).

    CAS  Article  Google Scholar 

  6. 6.

    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).

    CAS  Article  Google Scholar 

  7. 7.

    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).

    CAS  Article  Google Scholar 

  8. 8.

    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).

    CAS  Article  Google Scholar 

  9. 9.

    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).

    CAS  Article  Google Scholar 

  10. 10.

    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).

    CAS  Article  Google Scholar 

  11. 11.

    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).

    CAS  Article  Google Scholar 

  12. 12.

    Woolfson, D. N. The design of coiled-coil structures and assemblies. Adv. Protein Chem. 70, 79–112 (2005).

    CAS  Article  Google Scholar 

  13. 13.

    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).

    CAS  Article  Google Scholar 

  14. 14.

    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).

    CAS  Article  Google Scholar 

  15. 15.

    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).

    Article  Google Scholar 

  16. 16.

    Chen, Z. et al. Programmable design of orthogonal protein heterodimers. Nature 565, 106–111 (2019).

    CAS  Article  Google Scholar 

  17. 17.

    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).

    CAS  Article  Google Scholar 

  18. 18.

    Gradišar, H. & Jerala, R. De novo design of orthogonal peptide pairs forming parallel coiled-coil heterodimers. J. Pept. Sci. 17, 100–106 (2011).

    Article  Google Scholar 

  19. 19.

    Gradišar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat. Chem. Biol. 9, 362–366 (2013).

    Article  Google Scholar 

  20. 20.

    O’Shea, E. K., Lumb, K. J. & Kim, P. S. Peptide ‘Velcro’: design of a heterodimeric coiled coil. Curr. Biol. 3, 658–667 (1993).

    Article  Google Scholar 

  21. 21.

    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).

    CAS  Article  Google Scholar 

  22. 22.

    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).

    CAS  Article  Google Scholar 

  23. 23.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Gao, Y. et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat. Methods 13, 1043–1049 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Mansouri, M., Strittmatter, T. & Fussenegger, M. Light-controlled mammalian cells and their therapeutic applications in synthetic biology. Adv. Sci. 6, 1800952 (2019).

    Article  Google Scholar 

  27. 27.

    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).

    Article  Google Scholar 

  28. 28.

    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).

    CAS  Article  Google Scholar 

  29. 29.

    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).

    Article  Google Scholar 

  30. 30.

    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).

    Article  Google Scholar 

  31. 31.

    Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    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).

    Article  Google Scholar 

  33. 33.

    Weber, W. & Fussenegger, M. Emerging biomedical applications of synthetic biology. Nat. Rev. Genet. 13, 21–35 (2012).

    CAS  Article  Google Scholar 

  34. 34.

    Kemmer, C. et al. Self-sufficient control of urate homeostasis in mice by a synthetic circuit. Nat. Biotechnol. 28, 355–360 (2010).

    CAS  Article  Google Scholar 

  35. 35.

    Wasilewska, A. et al. An update on abscisic acid signaling in plants and Mor. Mol. Plant. 1, 198–217 (2008).

    CAS  Article  Google Scholar 

  36. 36.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  Article  Google Scholar 

  37. 37.

    Breuza, L. et al. The UniProtKB guide to the human proteome. Database 2016, bav120 (2016).

    Article  Google Scholar 

  38. 38.

    UniProt Consortium, T. U. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 47, D506–D515 (2019).

    Article  Google Scholar 

  39. 39.

    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).

    CAS  Article  Google Scholar 

  40. 40.

    Kwak, S. K. & Kim, J. H. Statistical data preparation: management of missing values and outliers. Korean J. Anesthesiol. 70, 407–411 (2017).

    Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

T.L. and E.M. prepared the plasmid constructs and performed the experiments on cell culture. D.L. performed the experiments on mice. J.A. performed the bioinformatics analysis. T.L. and R.J. designed and analyzed the experiments and wrote the manuscript. R.J. conceived the study.

Corresponding author

Correspondence to Roman Jerala.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary information

Supplementary Tables 1–6 and Figs. 1–16

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lebar, T., Lainšček, D., Merljak, E. et al. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat Chem Biol 16, 513–519 (2020). https://doi.org/10.1038/s41589-019-0443-y

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing