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

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, s

SUBMITTER: Scheele RA 

PROVIDER: S-EPMC8837617 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature