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A multiplexed bacterial two-hybrid for rapid characterization of protein–protein interactions and iterative protein design

Author

Listed:
  • W. Clifford Boldridge

    (University of California)

  • Ajasja Ljubetič

    (National Institute of Chemistry
    EN-FIST Centre of Excellence)

  • Hwangbeom Kim

    (University of California
    Samsung Biologics)

  • Nathan Lubock

    (University of California
    Octant Inc)

  • Dániel Szilágyi

    (University of Primorska)

  • Jonathan Lee

    (University of California
    University of Southern California)

  • Andrej Brodnik

    (University of Primorska)

  • Roman Jerala

    (National Institute of Chemistry
    EN-FIST Centre of Excellence)

  • Sriram Kosuri

    (University of California
    University of California, Los Angeles
    University of California, Los Angeles
    University of California, Los Angeles)

Abstract

Protein-protein interactions (PPIs) are crucial for biological functions and have applications ranging from drug design to synthetic cell circuits. Coiled-coils have been used as a model to study the sequence determinants of specificity. However, building well-behaved sets of orthogonal pairs of coiled-coils remains challenging due to inaccurate predictions of orthogonality and difficulties in testing at scale. To address this, we develop the next-generation bacterial two-hybrid (NGB2H) method, which allows for the rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way using next-generation sequencing readout. We design, build, and test large sets of orthogonal synthetic coiled-coils, assayed over 8,000 PPIs, and used the dataset to train a more accurate coiled-coil scoring algorithm (iCipa). After characterizing nearly 18,000 new PPIs, we identify to the best of our knowledge the largest set of orthogonal coiled-coils to date, with fifteen on-target interactions. Our approach provides a powerful tool for the design of orthogonal PPIs.

Suggested Citation

  • W. Clifford Boldridge & Ajasja Ljubetič & Hwangbeom Kim & Nathan Lubock & Dániel Szilágyi & Jonathan Lee & Andrej Brodnik & Roman Jerala & Sriram Kosuri, 2023. "A multiplexed bacterial two-hybrid for rapid characterization of protein–protein interactions and iterative protein design," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-38697-x
    DOI: 10.1038/s41467-023-38697-x
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    References listed on IDEAS

    as
    1. Po-Ssu Huang & Scott E. Boyken & David Baker, 2016. "The coming of age of de novo protein design," Nature, Nature, vol. 537(7620), pages 320-327, September.
    2. Jana Aupič & Žiga Strmšek & Fabio Lapenta & David Pahovnik & Tomaž Pisanski & Igor Drobnak & Ajasja Ljubetič & Roman Jerala, 2021. "Designed folding pathway of modular coiled-coil-based proteins," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    3. Jae-Seong Yang & Mireia Garriga-Canut & Nele Link & Carlo Carolis & Katrina Broadbent & Violeta Beltran-Sastre & Luis Serrano & Sebastian P. Maurer, 2018. "rec-YnH enables simultaneous many-by-many detection of direct protein–protein and protein–RNA interactions," Nature Communications, Nature, vol. 9(1), pages 1-13, December.
    4. Zibo Chen & Scott E. Boyken & Mengxuan Jia & Florian Busch & David Flores-Solis & Matthew J. Bick & Peilong Lu & Zachary L. VanAernum & Aniruddha Sahasrabuddhe & Robert A. Langan & Sherry Bermeo & T. , 2019. "Programmable design of orthogonal protein heterodimers," Nature, Nature, vol. 565(7737), pages 106-111, January.
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