Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates.
Lindenburg, Laurens H
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Scheele, R., 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.. Nat Commun, 13 (1) https://doi.org/10.1038/s41467-022-28396-4
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.
Humans, Phosphates, Protein Kinases, MAP Kinase Kinase 1, Mitogen-Activated Protein Kinases, Mitogen-Activated Protein Kinase 1, Signal Transduction, MAP Kinase Signaling System, Epistasis, Genetic, Substrate Specificity, Phosphorylation, Catalysis, Protein-Tyrosine Kinases, Molecular Docking Simulation, Protein Domains
RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) - BB/M011194/1 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 721613 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 659029 EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) - 695669
European Commission Horizon 2020 (H2020) ERC (695664)
European Commission (330978)
European Commission (659029)
Biotechnology and Biological Sciences Research Council (BB/M011194/1)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (721613)
External DOI: https://doi.org/10.1038/s41467-022-28396-4
This record's URL: https://www.repository.cam.ac.uk/handle/1810/335043
Attribution 4.0 International
Licence URL: https://creativecommons.org/licenses/by/4.0/