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.

Chemogenetic Control of Nanobodies

Abstract

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Generation of LAMAs from nanobodies and cpDHFR.
Fig. 2: Sequester and release of protein localization in live cells using LAMAs.

Similar content being viewed by others

Data availability

Plasmids encoding for LAMAs have been deposited on Addgene with accession codes 130704 to 130718 and 136618 to 136635. All requests for the Nup62-mEGFP genome-edited cell line should be directed to J.E. Structures of GFPLAMAF98 and GFPLAMAG97 have been deposited to the PDB with deposition codes 6RUL and 6RUM, respectively. The source data for Figs. 1c–g,j,l and 2e,f,j,k are provided with the paper online. Additional datasets that support the finding of this study are available from the corresponding author upon request.

References

  1. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).

    Article  CAS  PubMed  Google Scholar 

  2. Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Ingram, J. R., Schmidt, F. I. & Ploegh, H. L. Exploiting nanobodies’ singular traits. Annu. Rev. Immunol. 36, 695–715 (2018).

    Article  CAS  PubMed  Google Scholar 

  4. Stein, V. & Alexandrov, K. Synthetic protein switches: design principles and applications. Trends Biotechnol. 33, 101–110 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Oakes, B. L. et al. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nat. Biotechnol. 34, 646–651 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V. & Hahn, K. M. Engineered allosteric activation of kinases in living cells. Nat. Biotechnol. 28, 743 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gil, A. A. et al. Optogenetic control of protein binding using light-switchable nanobodies. Preprint at bioRxiv https://doi.org/10.1101/739201 (2019).

  8. Yu, D. et al. Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins. Nat. Methods 16, 1095–1100 (2019).

    Article  CAS  PubMed  Google Scholar 

  9. Iwakura, M. & Nakamura, T. Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase. Protein Eng. 11, 707–713 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Yu, Q. et al. Semisynthetic sensor proteins enable metabolic assays at the point of care. Science 361, 1122–1126 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. Nakamura, T. & Iwakura, M. Circular permutation analysis as a method for distinction of functional elements in the M20 loop of Escherichia colidihydrofolate reductase. J. Biol. Chem. 274, 19041–19047 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. De Genst, E. et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc. Natl Acad. Sci. USA 103, 4586–4591 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wilton, E. E., Opyr, M. P., Kailasam, S., Kothe, R. F. & Wieden, H.-J. sdAb-DB: the single domain antibody database. ACS Synth. Biol. 7, 2480–2484 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Chaikuad, A. et al. Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies. Biochem. J. 459, 59–69 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Sosa, B. A. et al. How lamina-associated polypeptide 1 (LAP1) activates torsin. Elife 3, e03239 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Götzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 10, 4403 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tao, R. et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism. Nat. Methods 14, 720–728 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kanaji, S., Iwahashi, J., Kida, Y., Sakaguchi, M. & Mihara, K. Characterization of the signal that directs Tom20 to the mitochondrial outer membrane. J. Cell Biol. 151, 277–288 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Inoue, T., Heo, W. D., Grimley, J. S., Wandless, T. J. & Meyer, T. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat. Methods 2, 415–418 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Murakoshi, H., Shibata, A. C. E., Nakahata, Y. & Nabekura, J. A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer. Sci. Rep. 5, 15334 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Held, M. et al. CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nat. Methods 7, 747–754 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Cai, Y. et al. Experimental and computational framework for a dynamic protein atlas of human cell division. Nature 561, 411–415 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Santaguida, S., Tighe, A., Alise, A. M., Taylor, S. S. & Musacchio, A. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J. Cell Biol. 190, 73–87 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Müller, B. et al. Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J. Virol. 78, 10803–10813 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lampe, M. et al. Double-labelled HIV-1 particles for study of virus–cell interaction. Virology 360, 92–104 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Hendrix, J. et al. Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers. J. Cell Biol. 210, 629–646 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Trotard, M. et al. Sensing of HIV-1 infection in Tzm-bl cells with reconstituted expression of STING. J. Virol. 90, 2064–2076 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Keppler, A., Pick, H., Arrivoli, C., Vogel, H. & Johnsson, K. Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl Acad. Sci. USA 101, 9955–9959 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Roehrl, M. H. A., Wang, J. Y. & Wagner, G. A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein−protein interactions by fluorescence polarization. Biochemistry 43, 16056–16066 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Cabrita, L. D. et al. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16, 2360–2367 (2009).

    Article  CAS  Google Scholar 

  34. Kabsch, W. XDS. Acta Crystallogr. D. Struct. Biol. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  35. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. Struct. Biol. 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  38. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. DeLano, W. L. Pymol: an open-source molecular graphics tool. CCP4 newsletter on protein. Crystallography 40, 82–92 (2002).

    Google Scholar 

  41. Koch, B. et al. Generation and validation of homozygous fluorescent knock-in cells using CRISPR–Cas9 genome editing. Nat. Protoc. 13, 1465–1487 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Otsuka, S. et al. Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings. Nat. Struct. Mol. Biol. 25, 21–28 (2018).

    Article  CAS  PubMed  Google Scholar 

  43. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Max Planck Society, the École Polytechnique Fédérale de Lausanne and NCCR Chemical Biology. Research in Kräusslich’s group was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (Projektnummer 240245660) SFB 1129 project 5 (H.-G.K). Research in Ellenberg’s group was supported by the Paul G. Allen Frontiers Group through an Allen Distinguished Investigators Grant to J.E., the National Institutes of Health Common Fund 4D Nucleome Program (grant no. U01 EB021223/U01 DA047728 to J.E.) and the EMBL (S.O., M.K. and J.E.). We thank I. Schlichting for X-ray data collection. Diffraction data were collected at the Swiss Light Source, beamline X10SA, of the Paul Scherrer Institute, Villigen, Switzerland. We thank L. Reymond, J. Broichhagen, B. Mathes and A. Bergner for providing reagents and M. Eguren for valuable discussions.

Author information

Authors and Affiliations

Authors

Contributions

H.F. and K.J. designed the study. H.F generated, characterized and applied all LAMAs. M.T. solved the crystal structures of GFPLAMAs. J.H. helped analyze the crystal structures. M.K. generated the NUP62-mEGFP cell line and S.O. performed the NUP62-mEGFP translocation experiments. B.K. helped with generation of stable cell lines with LAMAs. T.G.M. generated stable cells lines of p24LAMA and characterized them. H.-G.K., J.E. and K.J. supervised the work. H.F and K.J. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Kai Johnsson.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–19 and Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Reversibility of mito-GFPLAMAF98 with EGFP in live cells; HeLa Kyoto cells expressing mito-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:19 min s. Complete media was perfused over the cells at 23:24–33:42 min s. TMP was again perfused over the cells at 39:49–49:51 min s. Complete media was again perfused over the cells at 00:55:56–1:08:05 h min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 2

Reversibility of mito-GFPLAMAG97 with EGFP in live cells. HeLa Kyoto cells expressing mito-GFPLAMAG97 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:19 min s. Complete media was perfused over the cells at 48:01–58:03 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 3

Reversibility of Lyn-GFPLAMAF98 with EGFP in live cells. HeLa Kyoto cells expressing Lyn-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–15:30 min s. Complete media was perfused over the cells at 20:40–28:34 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 4

Reversibility of nuc-GFPLAMAF98 with EGFP in live cells. HeLa Kyoto cells expressing nuc-GFPLAMAF98 IRES EGFP. TMP (10 µM) in complete media was perfused over the cells at 3:38–17:01 min s. Complete media was perfused over the cells at 22:11–31:36 min s. Fluorescence of EGFP was imaged (green). Scale bar, 10 µm.

Supplementary Video 5

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-GFPLAMAF98 in the absence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tagged mito-GFPLAMAF98. TMP (50 µM) was washed out at time point 0. Nucleus (blue), mito-GFPLAMAF98 (magenta) and transmission image (gray). Scale bar, 20 µm.

Supplementary Video 6

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-GFPLAMAF98 in the presence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tagged mito-GFPLAMAF98 and kept in TMP (50 µM) during imaging. Nucleus (blue), mito-GFPLAMAF98 (magenta) and transmission image (gray). Scale bar, 20 µm.

Supplementary Video 7

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-SNAP-tag in the absence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tag on the outer membrane of the mitochondria. TMP (50 µM) was washed out at time point 0. Nucleus (blue), mito-SNAP-tag (magenta) and transmission image (gray). Scale bar, 20 µm.

Supplementary Video 8

Mitosis in a genome-edited Mad2L1-EGFP cell line with mito-SNAP-tag in the presence of TMP. Genome-edited Mad2L1-EGFP cells stably expressing SNAP-tag on the outer membrane of the mitochondria and kept in TMP (50 µM) during imaging. Nucleus (blue), mitoSNAP-tag (magenta) and transmission image (gray). Scale bar, 20 µm.

Source data

Source Data Fig. 1

Statistical source data

Source Data Fig. 2

Statistical source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Farrants, H., Tarnawski, M., Müller, T.G. et al. Chemogenetic Control of Nanobodies. Nat Methods 17, 279–282 (2020). https://doi.org/10.1038/s41592-020-0746-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-020-0746-7

This article is cited by

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