De novo design of protein logic gates

Creators: Chen, Zibo; Kibler, Ryan D.; Hunt, Andrew; Busch, Florian; Pearl, Jocelynn; Jia, Mengxuan; VanAernum, Zachary L.; Wicky, Basile I. M.; Dods, Galen; Liao, Hanna; Wilken, Matthew S.; Ciarlo, Christie; Green, Shon; El-Samad, Hana; Stamatoyannopoulos, John; Wysocki, Vicki H.; Jewett, Michael C.; Boyken, Scott E.; Baker, David

Abstract

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo–designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.

Additional Information

© 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. This is an article distributed under the terms of the Science Journals Default License. Received for publication June 5, 2019. Accepted for publication March 5, 2020. We thank A. Ng, T. Nguyen, D. Younger, M. Xie, and B. Groves for assistance with yeast experiments; C. Chow for assistance with protein purification; W. Lim for discussions on protein-binding cooperativity; M. Elowitz, R. Schulman, and N. Woodall for feedback on the manuscript; S. Bermeo, M. Lajoie, R. Langan, and A. Ljubetič for useful discussions; and S. Pennington for making the media for Y2H assays. Funding: This work was supported by the Howard Hughes Medical Institute (D.B.), the generosity of Eric and Wendy Schmidt by recommendation of the Schmidt Futures program (D.B. and Z.C.), IPD-WA State funding Y5/07-5568 (D.B.), NIH BTRR Yeast Resource Grant Y8-12/61-3650 (Z.C. and R.D.K.), Bruce and Jeannie Nordstrom/Patty and Jimmy Barrier Gift for the Institute for Protein Design (Z.C. and R.D.K.), Spark ABCA4/63-3819 (Z.C.), NIH P41 grant GM103533 (R.D.K.), Open Philanthropy (D.B. and B.I.M.W.), EMBO/80-7223 (B.I.M.W.), Burroughs Wellcome Fund Career Award at the Scientific Interface (S.E.B.), the Army Research Office W911NF-18-1-0200 (M.C.J.), the Air Force Research Laboratory Center of Excellence grant FA8650-15-2-5518 (M.C.J), the David and Lucile Packard Foundation (M.C.J.), and the Camille Dreyfus Teacher-Scholar Program (M.C.J.). A.H. was supported by the Department of Defense (DOD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program. T cell engineering work was supported by a charitable contribution from GlaxoSmithKline to the Altius Institute for Biomedical Sciences (J.R.P., H.L., M.W., C.C., J.A.S.) and NHLBI grant 7R33HL120752 (J.A.S). V.H.W. was supported by NIH grant P41 GM128577 and Ohio Eminent Scholar funds. H.E.-S. was supported by the Defense Advanced Research Projects Agency, contract no. HR0011-16-2-0045, and is a Chan-Zuckerberg investigator. SAXS data were collected at the Advanced Light Source (ALS) at LBNL, supported by the following grants from the NIH: P30 GM124169-01, ALS-ENABLE P30 GM124169, and S10OD018483; NCI SBDR (CA92584); and DOE-BER IDAT (DE-AC02-05CH11231). Author contributions: Z.C., S.E.B., and D.B. conceived of the idea; Z.C. and D.B. designed the research; Z.C. and D.B. wrote the manuscript with help from R.D.K and S.E.B.; Z.C. and R.D.K. performed biophysical characterizations with help from B.I.M.W.; Z.C. performed Y2H experiments; A.H. performed NanoBiT experiments and analyzed data with M.C.J.; F.B., M.J., and Z.L.V. performed nMS experiments and analyzed data with V.H.W.; J.R.P., M.W., and C.C. performed and analyzed data from T cell experiments with transcriptional repressors, supervised by S.G. and J.A.S.; G.D. performed the yeast titration assay and analyzed data with H.E.-S.; all authors discussed the results and commented on the manuscript. Competing interests: Z.C, S.E.B., and D.B. are coinventors on patent application PCT/US19/59654 that incorporates discoveries described in this manuscript. D.B. is a cofounder of and holds equity in Lyell Immunopharma and Sana Biotechnology. J.P., M.W., C.C., S.G., and J.A.S. are co-inventors on U.S. patent application 62/937,011 that includes discoveries described in this manuscript. J.P. H.L., and S.E.B. hold equity in Lyell Immunopharma. Data and materials availability: Raw data from nMS experiments have been deposited to http://files.ipd.uw.edu/pub/de_novo_logic_2019/190522_native_ms_raw.zip. Code used to simulate the cooperative binding system has been deposited at Zenodo (52). Plasmids used in this study are available upon request from the corresponding author.

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