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

Remkes A. Scheele, Laurens H. Lindenburg, Maya Petek, Markus Schober, Kevin N. Dalby and Florian Hollfelder ()
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Remkes A. Scheele: University of Cambridge
Laurens H. Lindenburg: University of Cambridge
Maya Petek: University of Cambridge
Markus Schober: University of Cambridge
Kevin N. Dalby: The University of Texas at Austin
Florian Hollfelder: University of Cambridge

Nature Communications, 2022, vol. 13, issue 1, 1-14

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-28396-4

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