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Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers

Ubiquitylation is an eminent posttranslational modification referring to the covalent attachment of single ubiquitin molecules or polyubiquitin chains to a target protein dictating the fate of such labeled polypeptide chains. Here, we have biochemically produced artificially Lys11-, and Lys27-, and...

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Autores principales: Schneider, Tobias, Berg, Andrej, Ulusoy, Zeynel, Gamerdinger, Martin, Peter, Christine, Kovermann, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934565/
https://www.ncbi.nlm.nih.gov/pubmed/31882959
http://dx.doi.org/10.1038/s41598-019-56458-z
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author Schneider, Tobias
Berg, Andrej
Ulusoy, Zeynel
Gamerdinger, Martin
Peter, Christine
Kovermann, Michael
author_facet Schneider, Tobias
Berg, Andrej
Ulusoy, Zeynel
Gamerdinger, Martin
Peter, Christine
Kovermann, Michael
author_sort Schneider, Tobias
collection PubMed
description Ubiquitylation is an eminent posttranslational modification referring to the covalent attachment of single ubiquitin molecules or polyubiquitin chains to a target protein dictating the fate of such labeled polypeptide chains. Here, we have biochemically produced artificially Lys11-, and Lys27-, and Lys63-linked ubiquitin dimers based on click-chemistry generating milligram quantities in high purity. We show that the artificial linkage used for the conjugation of two ubiquitin moieties represents a fully reliable surrogate of the natural isopeptide bond by acquiring highly resolved nuclear magnetic resonance (NMR) spectroscopic data including ligand binding studies. Extensive coarse grained and atomistic molecular dynamics (MD) simulations allow to extract structures representing the ensemble of domain-domain conformations used to verify the experimental data. Advantageously, this methodology does not require individual isotopic labeling of both ubiquitin moieties as NMR data have been acquired on the isotopically labeled proximal moiety and complementary MD simulations have been used to fully interpret the experimental data in terms of domain-domain conformation. This combined approach intertwining NMR spectroscopy with MD simulations makes it possible to describe the conformational space non-canonically Lys11-, and Lys27-linked ubiquitin dimers occupy in a solution averaged ensemble by taking atomically resolved information representing all residues in ubiquitin dimers into account.
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spelling pubmed-69345652019-12-29 Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers Schneider, Tobias Berg, Andrej Ulusoy, Zeynel Gamerdinger, Martin Peter, Christine Kovermann, Michael Sci Rep Article Ubiquitylation is an eminent posttranslational modification referring to the covalent attachment of single ubiquitin molecules or polyubiquitin chains to a target protein dictating the fate of such labeled polypeptide chains. Here, we have biochemically produced artificially Lys11-, and Lys27-, and Lys63-linked ubiquitin dimers based on click-chemistry generating milligram quantities in high purity. We show that the artificial linkage used for the conjugation of two ubiquitin moieties represents a fully reliable surrogate of the natural isopeptide bond by acquiring highly resolved nuclear magnetic resonance (NMR) spectroscopic data including ligand binding studies. Extensive coarse grained and atomistic molecular dynamics (MD) simulations allow to extract structures representing the ensemble of domain-domain conformations used to verify the experimental data. Advantageously, this methodology does not require individual isotopic labeling of both ubiquitin moieties as NMR data have been acquired on the isotopically labeled proximal moiety and complementary MD simulations have been used to fully interpret the experimental data in terms of domain-domain conformation. This combined approach intertwining NMR spectroscopy with MD simulations makes it possible to describe the conformational space non-canonically Lys11-, and Lys27-linked ubiquitin dimers occupy in a solution averaged ensemble by taking atomically resolved information representing all residues in ubiquitin dimers into account. Nature Publishing Group UK 2019-12-27 /pmc/articles/PMC6934565/ /pubmed/31882959 http://dx.doi.org/10.1038/s41598-019-56458-z Text en © The Author(s) 2019 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/.
spellingShingle Article
Schneider, Tobias
Berg, Andrej
Ulusoy, Zeynel
Gamerdinger, Martin
Peter, Christine
Kovermann, Michael
Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
title Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
title_full Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
title_fullStr Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
title_full_unstemmed Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
title_short Conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
title_sort conformational and functional characterization of artificially conjugated non-canonical ubiquitin dimers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934565/
https://www.ncbi.nlm.nih.gov/pubmed/31882959
http://dx.doi.org/10.1038/s41598-019-56458-z
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