| Title: | Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates |
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| Authors: | ID Scheele, Remkes A. (Author)
ID Lindenburg, Laurens H. (Author)
ID Petek, Maya (Author)
ID Schober, Markus (Author)
ID Dalby, Kevin N. (Author)
ID Hollfelder, Florian (Author) |
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| Files: |  Scheele-2022-Droplet-based_screening_of_phosph.pdf (2,42 MB)
MD5: A49086104F829195C0E5737E82F4F3E1
 https://doi.org/10.1038/s41467-022-28396-4
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| Language: | English |
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| Work type: | Scientific work |
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| Typology: | 1.01 - Original Scientific Article |
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| Organization: | MF - Faculty of Medicine
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| 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. |
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| Keywords: | protein, epistasis, catalysis, phosphate transfer, MAP kinase |
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| Publication status: | Published |
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| Publication version: | Version of Record |
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| Submitted for review: | 07.04.2021 |
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| Article acceptance date: | 10.01.2022 |
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| Publication date: | 11.02.2022 |
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| Publisher: | Nature Publishing Group |
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| Year of publishing: | 2022 |
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| Number of pages: | Str. 1-14 |
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| Numbering: | Letn. 13, Št. članka 844 |
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| PID: | 20.500.12556/DKUM-89347  |
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| UDC: | 577 |
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| ISSN on article: | 2041-1723 |
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| COBISS.SI-ID: | 98894851 |
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| DOI: | 10.1038/s41467-022-28396-4 |
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| Publication date in DKUM: | 04.07.2024 |
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| Views: | 257 |
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| Downloads: | 20 |
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| Metadata: |    |
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| Categories: | Misc.
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