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Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates

Author

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  • Remkes A. Scheele

    (University of Cambridge)

  • Laurens H. Lindenburg

    (University of Cambridge)

  • Maya Petek

    (University of Cambridge
    University of Maribor)

  • Markus Schober

    (University of Cambridge)

  • Kevin N. Dalby

    (The University of Texas at Austin)

  • Florian Hollfelder

    (University of Cambridge)

Abstract

The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.

Suggested Citation

  • Remkes A. Scheele & Laurens H. Lindenburg & Maya Petek & Markus Schober & Kevin N. Dalby & Florian Hollfelder, 2022. "Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28396-4
    DOI: 10.1038/s41467-022-28396-4
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    References listed on IDEAS

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    1. Conor J. McClune & Aurora Alvarez-Buylla & Christopher A. Voigt & Michael T. Laub, 2019. "Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space," Nature, Nature, vol. 574(7780), pages 702-706, October.
    2. Frank J. Poelwijk & Michael Socolich & Rama Ranganathan, 2019. "Learning the pattern of epistasis linking genotype and phenotype in a protein," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    3. Javin P. Oza & Hans R. Aerni & Natasha L. Pirman & Karl W. Barber & Charlotte M. ter Haar & Svetlana Rogulina & Matthew B. Amrofell & Farren J. Isaacs & Jesse Rinehart & Michael C. Jewett, 2015. "Robust production of recombinant phosphoproteins using cell-free protein synthesis," Nature Communications, Nature, vol. 6(1), pages 1-7, November.
    4. Stephane Emond & Maya Petek & Emily J. Kay & Brennen Heames & Sean R. A. Devenish & Nobuhiko Tokuriki & Florian Hollfelder, 2020. "Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis," Nature Communications, Nature, vol. 11(1), pages 1-14, December.
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    Cited by:

    1. Philipp Knyphausen & Mariana Rangel Pereira & Paul Brear & Marko Hyvönen & Lutz Jermutus & Florian Hollfelder, 2023. "Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture," Nature Communications, Nature, vol. 14(1), pages 1-13, December.

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