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

Published version
Peer-reviewed

Change log

Authors

Scheele, Remkes A 
Lindenburg, Laurens H 
Schober, Markus 

Abstract

jats:titleAbstract</jats:title>jats:pThe 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 (>10jats:sup7</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>

Description

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

Journal Title

Nature Communications

Conference Name

Journal ISSN

2041-1723

Volume Title

13

Publisher

Springer Science and Business Media LLC
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)