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.
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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.
Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993).
Muyldermans, S. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82, 775–797 (2013).
Ingram, J. R., Schmidt, F. I. & Ploegh, H. L. Exploiting nanobodies’ singular traits. Annu. Rev. Immunol. 36, 695–715 (2018).
Stein, V. & Alexandrov, K. Synthetic protein switches: design principles and applications. Trends Biotechnol. 33, 101–110 (2015).
Oakes, B. L. et al. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch. Nat. Biotechnol. 34, 646–651 (2016).
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).
Gil, A. A. et al. Optogenetic control of protein binding using light-switchable nanobodies. Preprint at bioRxiv https://doi.org/10.1101/739201 (2019).
Yu, D. et al. Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins. Nat. Methods 16, 1095–1100 (2019).
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).
Yu, Q. et al. Semisynthetic sensor proteins enable metabolic assays at the point of care. Science 361, 1122–1126 (2018).
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).
Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2009).
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).
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).
Chaikuad, A. et al. Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies. Biochem. J. 459, 59–69 (2014).
Sosa, B. A. et al. How lamina-associated polypeptide 1 (LAP1) activates torsin. Elife 3, e03239 (2014).
Götzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 10, 4403 (2019).
Tao, R. et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism. Nat. Methods 14, 720–728 (2017).
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).
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).
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).
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).
Held, M. et al. CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging. Nat. Methods 7, 747–754 (2010).
Cai, Y. et al. Experimental and computational framework for a dynamic protein atlas of human cell division. Nature 561, 411–415 (2018).
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).
Müller, B. et al. Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J. Virol. 78, 10803–10813 (2004).
Lampe, M. et al. Double-labelled HIV-1 particles for study of virus–cell interaction. Virology 360, 92–104 (2007).
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).
Trotard, M. et al. Sensing of HIV-1 infection in Tzm-bl cells with reconstituted expression of STING. J. Virol. 90, 2064–2076 (2016).
Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).
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).
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).
Cabrita, L. D. et al. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16, 2360–2367 (2009).
Kabsch, W. XDS. Acta Crystallogr. D. Struct. Biol. 66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).
Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. Struct. Biol. 67, 355–367 (2011).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D. Biol. Crystallogr. 66, 12–21 (2010).
DeLano, W. L. Pymol: an open-source molecular graphics tool. CCP4 newsletter on protein. Crystallography 40, 82–92 (2002).
Koch, B. et al. Generation and validation of homozygous fluorescent knock-in cells using CRISPR–Cas9 genome editing. Nat. Protoc. 13, 1465–1487 (2018).
Otsuka, S. et al. Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings. Nat. Struct. Mol. Biol. 25, 21–28 (2018).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
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.
The authors declare no competing interests.
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 Figs. 1–19 and Tables 1 and 2.
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.
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.
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.
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.
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.
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.
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.
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.
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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
Nature Chemical Biology (2021)
Nature Methods (2020)
Nature Communications (2020)