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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 (>10(7) var...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8837617/ https://www.ncbi.nlm.nih.gov/pubmed/35149678 http://dx.doi.org/10.1038/s41467-022-28396-4 |
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author | Scheele, Remkes A. Lindenburg, Laurens H. Petek, Maya Schober, Markus Dalby, Kevin N. Hollfelder, Florian |
author_facet | Scheele, Remkes A. Lindenburg, Laurens H. Petek, Maya Schober, Markus Dalby, Kevin N. Hollfelder, Florian |
author_sort | Scheele, Remkes A. |
collection | PubMed |
description | 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 (>10(7) 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. |
format | Online Article Text |
id | pubmed-8837617 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-88376172022-03-04 Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates Scheele, Remkes A. Lindenburg, Laurens H. Petek, Maya Schober, Markus Dalby, Kevin N. Hollfelder, Florian Nat Commun Article 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 (>10(7) 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. Nature Publishing Group UK 2022-02-11 /pmc/articles/PMC8837617/ /pubmed/35149678 http://dx.doi.org/10.1038/s41467-022-28396-4 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Scheele, Remkes A. Lindenburg, Laurens H. Petek, Maya Schober, Markus Dalby, Kevin N. Hollfelder, Florian Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates |
title | Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates |
title_full | Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates |
title_fullStr | Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates |
title_full_unstemmed | Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates |
title_short | Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates |
title_sort | droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes map kinase interactions with substrates |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8837617/ https://www.ncbi.nlm.nih.gov/pubmed/35149678 http://dx.doi.org/10.1038/s41467-022-28396-4 |
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