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dc.contributor.authorScheele, Remkes
dc.contributor.authorLindenburg, Laurens H
dc.contributor.authorPetek, Maya
dc.contributor.authorSchober, Markus
dc.contributor.authorDalby, Kevin N
dc.contributor.authorHollfelder, Florian
dc.date.accessioned2022-01-13T00:31:08Z
dc.date.available2022-01-13T00:31:08Z
dc.date.issued2022-02-11
dc.identifier.issn2041-1723
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/332670
dc.description.abstractThe 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.
dc.description.sponsorshipRCUK | Biotechnology and Biological Sciences Research Council (BBSRC) - BB/M011194/1 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 721613 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 659029 EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) - 695669
dc.publisherNature Research
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.titleDroplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates.
dc.typeArticle
dc.publisher.departmentDepartment of Biochemistry
dc.date.updated2022-01-10T22:31:35Z
prism.publicationNameNat Commun
dc.identifier.doi10.17863/CAM.80115
dcterms.dateAccepted2022-01-10
rioxxterms.versionofrecord10.1038/s41467-022-28396-4
rioxxterms.versionAM
dc.contributor.orcidPetek, Maya [0000-0002-4597-4162]
dc.contributor.orcidDalby, Kevin N [0000-0001-9272-5129]
dc.contributor.orcidHollfelder, Florian [0000-0002-1367-6312]
dc.identifier.eissn2041-1723
rioxxterms.typeJournal Article/Review
pubs.funder-project-idEuropean Commission Horizon 2020 (H2020) ERC (695664)
pubs.funder-project-idEuropean Commission (330978)
pubs.funder-project-idEuropean Commission (659029)
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (BB/M011194/1)
pubs.funder-project-idEuropean Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (721613)
cam.orpheus.successThu Feb 24 18:06:39 GMT 2022 - Embargo updated*
cam.orpheus.counter1
cam.depositDate2022-01-10
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement
rioxxterms.freetoread.startdate2022-02-11


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International