20.500.12556/DKUM-89347 Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates 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. protein epistasis catalysis phosphate transfer MAP kinase proteini epistaza kataliza prenos fosfatov MAP-kinaza true false true Nature Publishing Group Angleški jezik Slovenski jezik Znanstveno delo 2024-07-04 08:55:41 2024-07-04 09:16:12 2024-07-05 04:13:28 0000-00-00 00:00:00 2022 0 0 Str. 1-14 Št. članka 844 Letn. 13 2021 0000-00-00 Zaloznikova Objavljeno NiDoloceno 2021-04-07 2022-01-10 2022-02-11 577 98894851 10.1038/s41467-022-28396-4 2041-1723 Scheele-2022-Droplet-based_screening_of_phosph.pdf Scheele-2022-Droplet-based_screening_of_phosph.pdf 1 A49086104F829195C0E5737E82F4F3E1 529f83e441753d5210597ed9fe0dd6b1c38ff222dc85d79fe95dbd7939e30e93 069a7969-39d5-11ef-8f49-00155d000105 https://dk.um.si/Dokument.php?lang=slv&id=181106 https://doi.org/10.1038/s41467-022-28396-4 0 72c0af43-39d2-11ef-8f49-00155d000105 https://dk.um.si/Dokument.php?lang=slv&id=181105 Medicinska fakulteta 0 0 0