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Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins


Nowak, Kathrin; Rosenthal, Florian; Karlberg, Tobias; Bütepage, Mareike; Thorsell, Ann-Gerd; Dreier, Birgit; Grossmann, Jonas; Sobek, Jens; Imhof, Ralph; Lüscher, Bernhard; Schüler, Herwig; Plückthun, Andreas; Leslie Pedrioli, Deena M; Hottiger, Michael O (2020). Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins. Nature Communications, 11:5199.

Abstract

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

Abstract

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry

05 Vetsuisse Faculty > Veterinärwissenschaftliches Institut > Department of Molecular Mechanisms of Disease
07 Faculty of Science > Department of Molecular Mechanisms of Disease
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Physical Sciences > General Chemistry
Life Sciences > General Biochemistry, Genetics and Molecular Biology
Physical Sciences > General Physics and Astronomy
Language:English
Date:15 October 2020
Deposited On:18 Nov 2020 16:08
Last Modified:23 Jun 2024 01:45
Publisher:Nature Publishing Group
ISSN:2041-1723
OA Status:Gold
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1038/s41467-020-18981-w
PubMed ID:33060572
  • Content: Published Version
  • Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)