Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates
Authors
Scheele, Remkes A
Lindenburg, Laurens H
Schober, Markus
Publication Date
2022-12Journal Title
Nature Communications
Publisher
Springer Science and Business Media LLC
Volume
13
Issue
1
Language
en
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Scheele, R. A., Lindenburg, L. H., Petek, M., Schober, M., Dalby, K. N., & Hollfelder, F. (2022). Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates. Nature Communications, 13 (1) https://doi.org/10.1038/s41467-022-28396-4
Abstract
<jats:title>Abstract</jats:title><jats:p>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 (>10<jats:sup>7</jats:sup> 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.</jats:p>
Keywords
Article, /631/45/603, /631/45/275, /631/1647/2163, /631/92/605, /631/92/552, /49/31, /49/47, /49/23, /96/95, /96/35, /96/34, article
Sponsorship
EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) (721613, 659029, 330978, 721613)
EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) (695669, 695669)
RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) (BB/M011194/1, BB/M011194/1])
Welch Foundation (F-1390, F-1390)
Identifiers
s41467-022-28396-4, 28396
External DOI: https://doi.org/10.1038/s41467-022-28396-4
This record's URL: https://www.repository.cam.ac.uk/handle/1810/333925
Rights
Licence:
http://creativecommons.org/licenses/by/4.0/
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