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De novo design of tyrosine and serine kinase-driven protein switches

Abstract

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.

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Fig. 1: Phosphorylation switch design.
Fig. 2: Phosphorylation activates GFP fluorescence.
Fig. 3: Characterization of the phospho-states of pGFP-4Y.
Fig. 4: Serine-based phosphorylation switch.

Data availability

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.

Code availability

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.

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Acknowledgements

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.

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Authors

Contributions

D.B. and N.B.W. conceived the switch concept. N.B.W. and M.J.F. designed the switches. Z.W., J.P. and H.E.-S. characterized the serine switch in cells. M.M., M.A., N.B.W. and I.Y. characterized the tyrosine switch in cells. F.B. and V.H.W. characterized the tyrosine switch with native MS. R.S.J. and M.J.M. characterized the tyrosine switch with MS/MS. N.B.W. performed all other experiments. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to David Baker.

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Competing interests

N.W. and D.B. are inventors on US patent application PCT/US2020/038048. The remaining authors declare no competing interests.

Additional information

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 information

Supplementary Information

Supplementary Figs. 1–5 and Tables 1–4.

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Supplementary Data 1

Source data for Supplementary Fig. 5.

Supplementary Data 2

Protein sequences from the paper.

Supplementary Data 3

Design model for pGFP-4Y.

Supplementary Data 4

Design model for pGFP-4S.

Source data

Source Data Fig. 1

Source data.

Source Data Fig. 2

Source data.

Source Data Fig. 3

Source data.

Source Data Fig. 4

Source data.

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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

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