Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The PKA switch in vivo data are available at https://zenodo.org/record/5095560 and https://doi.org/10.5281/zenodo.5095560. Tryptic digest MS/MS data are available from the ProteomeXchange with identifier PXD027295. All protein sequences used in experiments are available as a Supplementary file, Protein_Sequences.xlsx. The design models experimentally tested are available as a supplementary protein databank file. The source backbone for the caged ITAM is available from the Protein Data Bank under ID 6DLC. The remainder of the experimental data and computational data are available at https://files.ipd.uw.edu/pub/PhosphateSwitch/DenovoPSwitch.zip. Unique biological materials (plasmids) are available upon request to the corresponding author. Source data are provided with this paper.
The custom Rosetta Scripts protocol for design with Rosetta 2018.19 is provided at https://github.com/NickWoodall/PhosphoSwitch_Design. Source code for PKA in vivo analysis is available at https://github.com/weinberz/phosphoswitch.
Balakrishnan, S. & Zondlo, N. J. Design of a protein kinase-inducible domain. J. Am. Chem. Soc. 128, 5590–5591 (2006).
Szilak, L., Moitra, J. & Vinson, C. Design of a leucine zipper coiled coil stabilized by 1.4 kcal mol−1 by phosphorylation of a serine in the e position. Protein Sci. 6, 1273–1283 (1997).
Smith, C. A. et al. Design of a phosphorylatable PDZ domain with peptide-specific affinity changes. Structure 21, 54–64 (2013).
Winter, D. L., Iranmanesh, H., Clark, D. S. & Glover, D. J. Design of tunable protein interfaces controlled by post-translational modifications. ACS Synth. Biol. 9, 2132–2143 (2020).
Schlessinger, J. & Ullrich, A. Growth factor signaling by receptor tyrosine kinases. Neuron 9, 383–391 (1992).
Hatada, M. et al. Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor. Nature 377, 32–38 (1995).
Langan, R. A. et al. De novo design of bioactive protein switches. Nature 572, 205–210 (2019).
Ng, A. H. et al. Modular and tunable biological feedback control using a de novo protein switch. Nature 572, 265–269 (2019).
Chen, Z. et al. Programmable design of orthogonal protein heterodimers. Nature 565, 106–111 (2019).
Shah, N., Lobel, M., Weiss, A. & Kuriyan, J. Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen. eLife 7, e35190 (2018).
Maguire, J. B., Boyken, S. E., Baker, D. & Kuhlman, B. Rapid sampling of hydrogen bond networks for computational protein design. J. Chem. Theory Comput. 14, 2751–2760 (2018).
Cabantous, S., Terwilliger, T. C. & Waldo, G. S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol. 23, 102–107 (2005).
Salazar, C. & Höfer, T. Multisite protein phosphorylation—from molecular mechanisms to kinetic models. FEBS J. 276, 3177–3198 (2009).
Mayer, B. J. Perspective: dynamics of receptor tyrosine kinase signaling complexes. FEBS Lett. 586, 2575–2579 (2012).
Dülk, M. et al. EGF regulates the interaction of Tks4 with Src through its SH2 and SH3 domains. Biochemistry 57, 4186–4196 (2018).
Hanna, R. A., Campbell, R. L. & Davies, P. L. Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin. Nature 456, 409–412 (2008).
Todd, B. et al. A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor. J. Mol. Biol. 328, 131–146 (2003).
Cuerrier, D. et al. Development of calpain-specific inactivators by screening of positional scanning epoxide libraries. J. Biol. Chem. 282, 9600–9611 (2007).
Robinson-White, A. & Stratakis, C. Protein kinase A signaling ‘cross-talk’ with other pathways in endocrine cells. Ann. NY Acad. Sci. 968, 256–270 (2002).
Kemp, B., Graves, D., Benjamini, E. & Krebs, E. Role of multiple basic residues in determining the substrate specifity of CAMP-dependent protein kinase. J. Biol. Chem. 252, 4888–4894 (1977).
Depry, C., Allen, M. D. & Zhang, J. Visualization of PKA activity in plasma membrane microdomains. Mol. Biosyst. 7, 52–58 (2011).
Chen, Y., Saulnier, J. L., Yellen, G. & Sabatini, B. L. A. PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. Front. Pharmacol 5, 56 (2014).
Mehta, S. et al. Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities. Nat. Cell Biol. 20, 1215–1225 (2018).
Iakoucheva, L. M. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 32, 1037–1049 (2004).
Torres, E. & Rosen, M. Contingent phosphorylation/dephosphorylation provides a mechanism of molecular memory in WASP. Mol. Cell 11, 1215–1227 (2003).
Kim, A., Kakalis, L., Abdul-Manan, N., Liu, G. & Rosen, M. Autoinhibition and activation mechanisms of the Wiskott–Aldrich syndrome protein. Nature 404, 151–158 (2000).
Antz, C. et al. NMR structure of inactivation gates from mammalian voltage-dependent potassium channels. Nature 385, 272–275 (1997).
Tsytlonok, M. et al. Dynamic anticipation by Cdk2/Cyclin A-bound p27 mediates signal integration in cell cycle regulation. Nat. Commun. 10, 1676 (2019).
Díaz-Moreno, I. et al. Phosphorylation-mediated unfolding of a KH domain regulates KSRP localization via 14-3-3 binding. Nat. Struct. Mol. Biol. 16, 238–246 (2009).
Antz, C. et al. Control of K. channel gating by protein phosphorylation: structural switches of the inactivation gate. Nat. Struct. Biol 6, 146–150 (1999).
Wilkins, J., Bitensky, M. & Willardson, B. Regulation of kinetics of phosducin phosphorylation in retinal rods. J. Biol. Chem. 271, 19232–19237 (1996).
Majzner, R. G. & Mackall, C. L. Clinical lessons learned from the first leg of the CAR T cell journey. Nat. Med. 25, 1341–1355 (2019).
Salter, A. et al. Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Sci. Signal. 11, eaat6753 (2018).
Sahoo, P. et al. Mathematical deconvolution of CAR T-cell proliferation and exhaustion from real-time killing assay data. J. R. Soc. Interface 17, 20190734 (2020).
Shin, H. et al. A role for the transcriptional repressor Blimp-1 in CD8+ T cell exhaustion during chronic viral infection. Immunity 31, 309–320 (2009).
Lu, P. et al. Blimp-1 represses CD8 T cell expression of PD-1 using a feed-forward transcriptional circuit during acute viral infection. J. Exp. Med. 211, 515–527 (2014).
Huang, P.-S. et al. RosettaRemodel: a generalized framework for flexible backbone protein design. PLoS ONE 6, e24109 (2011).
Fleishman, S. J. et al. RosettaScripts: a scripting language interface to the Rosetta Macromolecular Modeling Suite. PLoS ONE 6, e20161 (2011).
Bhardwaj, G. et al. Accurate de novo design of hyperstable constrained peptides. Nature 538, 329–335 (2016).
Hosseinzadeh, P. et al. Comprehensive computational design of ordered peptide macrocycles. Science 358, 1461–1466 (2017).
Albanese, S. K. et al. An open library of human kinase domain constructs for automated bacterial expression. Biochemistry 57, 4675–4689 (2018).
Zhang, Y. et al. Tail domains of myosin-1e regulate phosphatidylinositol signaling and F-actin polymerization at the ventral layer of podosomes. Mol. Biol. Cell 30, 622–635 (2019).
Kamiyama, D. et al. Versatile protein tagging in cells with split fluorescent protein. Nat. Commun. 7, 11046 (2016).
Boyken, S. E. et al. A conserved isoleucine maintains the inactive state of Bruton’s tyrosine kinase. J. Mol. Biol. 426, 3656–3669 (2014).
Deindl, S. et al. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70. Cell 129, 735–746 (2007).
Fonseca, P. et al. A toolkit for rapid modular construction of biological circuits in mammalian cells. ACS Synth. Biol 8, 2593–2606 (2019).
Filonov, G. S. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat. Biotechnol. 29, 757–761 (2011).
Graslund, S., Savitsky, P. & Muller-Knap, S. in Heterologous Gene Expression in E.coli Vol. 1586 (ed. Burgess-Brown, N. A.) 337–344 (Springer, 2017).
Wu, Z. et al. Impact of phosphorylation on the mass spectrometry quantification of intact phosphoproteins. Anal. Chem. 90, 4935–4939 (2018).
Dyer, K. N. et al. in Structural Genomics: General Applications (ed. Chen, Y. W.) 245–258 (Humana Press, 2014).
Rambo, R. P. & Tainer, J. A. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod–Debye law. Biopolymers 95, 559–571 (2011).
Schneidman-Duhovny, D., Hammel, M. & Sali, A. FoXS: a web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 38, W540–W544 (2010).
Schneidman-Duhovny, D., Hammel, M., Tainer, J. A. & Sali, A. Accurate SAXS profile computation and its assessment by contrast variation experiments. Biophys. J. 105, 962–974 (2013).
VanAernum, Z. L. et al. Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry. Nat. Protoc. 15, 1132–1157 (2020).
Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015).
Harris, C. R. et al. Array programming with NumPy. Nature 585, 357–362 (2020).
Virtanen, P. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17, 261–272 (2020).
N.B.W. and D.B. were supported by the Howard Hughes Medical Research Institute. M.M. was supported by The Audacious Project at the Institute for Protein Design. M.A. was supported by a gift from Amgen. I.Y. was supported by the NSF Graduate Research Fellowships Program (GRFP). Z.W. is supported by NIH 5K12GM081266. J.P. was supported by NIH 5T32GM007810. M.J.M. and R.S.J. are supported by NIGMS grant no. P41GM103533. D.B. and M.J.M. are supported by NIGMS U19 AG065156. Native mass spectrometry measurements were provided by the NIH-funded Resource for Native Mass Spectrometry Guided Structural Biology at The Ohio State University (NIH P41 GM128577 awarded to V.H.W.). We thank the staff at the Advanced Light Source SIBYLS beamline at Lawrence Berkeley National Laboratory, including K. Burnett, G. Hura, M. Hammel, J. Tanamachi and J. Tainer, for the services provided through the mail-in SAXS program, which is supported by the DOE Office of Biological and Environmental Research Integrated Diffraction Analysis program DOE BER IDAT grant (DE-AC02-05CH11231), NIGMS-supported ALS-ENABLE (GM124169-01) and National Institute of Health project MINOS (R01GM105404). Z.W. and J.P. thank A. Ng and A. Bonny, M. Kim for cloning advice and S. Allison for essential advice. We thank the Andreotti laboratory for their gift of the Lck kinase plasmid.
N.W. and D.B. are inventors on US patent application PCT/US2020/038048. The remaining authors declare no competing interests.
Peer review information Nature Structural & Molecular Biology thanks Dominic Glover and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Beth Moorefield 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. 1–5 and Tables 1–4.
Source data for Supplementary Fig. 5.
Protein sequences from the paper.
Design model for pGFP-4Y.
Design model for pGFP-4S.
About this article
Cite this article
Woodall, N.B., Weinberg, Z., Park, J. et al. De novo design of tyrosine and serine kinase-driven protein switches. Nat Struct Mol Biol 28, 762–770 (2021). https://doi.org/10.1038/s41594-021-00649-8