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De novo design of knotted tandem repeat proteins

Author

Listed:
  • Lindsey A. Doyle

    (Fred Hutchinson Cancer Center)

  • Brittany Takushi

    (Fred Hutchinson Cancer Center)

  • Ryan D. Kibler

    (University of Washington)

  • Lukas F. Milles

    (University of Washington)

  • Carolina T. Orozco

    (University of Cambridge, Lensfield Road)

  • Jonathan D. Jones

    (University of Cambridge, Lensfield Road)

  • Sophie E. Jackson

    (University of Cambridge, Lensfield Road)

  • Barry L. Stoddard

    (Fred Hutchinson Cancer Center)

  • Philip Bradley

    (Fred Hutchinson Cancer Center
    Fred Hutchinson Cancer Center)

Abstract

De novo protein design methods can create proteins with folds not yet seen in nature. These methods largely focus on optimizing the compatibility between the designed sequence and the intended conformation, without explicit consideration of protein folding pathways. Deeply knotted proteins, whose topologies may introduce substantial barriers to folding, thus represent an interesting test case for protein design. Here we report our attempts to design proteins with trefoil (31) and pentafoil (51) knotted topologies. We extended previously described algorithms for tandem repeat protein design in order to construct deeply knotted backbones and matching designed repeat sequences (N = 3 repeats for the trefoil and N = 5 for the pentafoil). We confirmed the intended conformation for the trefoil design by X ray crystallography, and we report here on this protein’s structure, stability, and folding behaviour. The pentafoil design misfolded into an asymmetric structure (despite a 5-fold symmetric sequence); two of the four repeat-repeat units matched the designed backbone while the other two diverged to form local contacts, leading to a trefoil rather than pentafoil knotted topology. Our results also provide insights into the folding of knotted proteins.

Suggested Citation

  • Lindsey A. Doyle & Brittany Takushi & Ryan D. Kibler & Lukas F. Milles & Carolina T. Orozco & Jonathan D. Jones & Sophie E. Jackson & Barry L. Stoddard & Philip Bradley, 2023. "De novo design of knotted tandem repeat proteins," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42388-y
    DOI: 10.1038/s41467-023-42388-y
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    References listed on IDEAS

    as
    1. Lindsey Doyle & Jazmine Hallinan & Jill Bolduc & Fabio Parmeggiani & David Baker & Barry L. Stoddard & Philip Bradley, 2015. "Rational design of α-helical tandem repeat proteins with closed architectures," Nature, Nature, vol. 528(7583), pages 585-588, December.
    2. Kathryn Tunyasuvunakool & Jonas Adler & Zachary Wu & Tim Green & Michal Zielinski & Augustin Žídek & Alex Bridgland & Andrew Cowie & Clemens Meyer & Agata Laydon & Sameer Velankar & Gerard J. Kleywegt, 2021. "Highly accurate protein structure prediction for the human proteome," Nature, Nature, vol. 596(7873), pages 590-596, August.
    3. William R. Taylor, 2000. "A deeply knotted protein structure and how it might fold," Nature, Nature, vol. 406(6798), pages 916-919, August.
    4. John Jumper & Richard Evans & Alexander Pritzel & Tim Green & Michael Figurnov & Olaf Ronneberger & Kathryn Tunyasuvunakool & Russ Bates & Augustin Žídek & Anna Potapenko & Alex Bridgland & Clemens Me, 2021. "Highly accurate protein structure prediction with AlphaFold," Nature, Nature, vol. 596(7873), pages 583-589, August.
    5. Nobuyasu Koga & Rie Tatsumi-Koga & Gaohua Liu & Rong Xiao & Thomas B. Acton & Gaetano T. Montelione & David Baker, 2012. "Principles for designing ideal protein structures," Nature, Nature, vol. 491(7423), pages 222-227, November.
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