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

Nowak, Kathrin ; Rosenthal, Florian ; Karlberg, Tobias LU ; Bütepage, Mareike ; Thorsell, Ann-Gerd ; Dreier, Birgit ; Grossmann, Jonas ; Sobek, Jens ; Imhof, Ralph and Lüscher, Bernhard , et al. (2020) In Nature Communications 11(1).
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... (More)

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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publishing date
type
Contribution to journal
publication status
published
keywords
ADP-Ribosylation/physiology, Adenosine Diphosphate Ribose/chemistry, Binding Sites, Carrier Proteins/genetics, HEK293 Cells, HeLa Cells, Humans, Models, Molecular, Mutagenesis, Protein Conformation, Protein Domains, Protein Engineering/methods, Protein Processing, Post-Translational, Proteins/chemistry, Proteomics/methods
in
Nature Communications
volume
11
issue
1
article number
5199
pages
14 pages
publisher
Nature Publishing Group
external identifiers
  • scopus:85092598386
  • pmid:33060572
ISSN
2041-1723
DOI
10.1038/s41467-020-18981-w
language
English
LU publication?
no
id
ce8d5502-f5cf-485a-8f2f-9d8b63f2a414
date added to LUP
2024-11-21 17:45:52
date last changed
2025-07-04 23:25:44
@article{ce8d5502-f5cf-485a-8f2f-9d8b63f2a414,
  abstract     = {{<p>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.</p>}},
  author       = {{Nowak, Kathrin and Rosenthal, Florian and Karlberg, Tobias and Bütepage, Mareike and Thorsell, Ann-Gerd and Dreier, Birgit and Grossmann, Jonas and Sobek, Jens and Imhof, Ralph and Lüscher, Bernhard and Schüler, Herwig and Plückthun, Andreas and Leslie Pedrioli, Deena M and Hottiger, Michael O}},
  issn         = {{2041-1723}},
  keywords     = {{ADP-Ribosylation/physiology; Adenosine Diphosphate Ribose/chemistry; Binding Sites; Carrier Proteins/genetics; HEK293 Cells; HeLa Cells; Humans; Models, Molecular; Mutagenesis; Protein Conformation; Protein Domains; Protein Engineering/methods; Protein Processing, Post-Translational; Proteins/chemistry; Proteomics/methods}},
  language     = {{eng}},
  month        = {{10}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Nature Communications}},
  title        = {{Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins}},
  url          = {{http://dx.doi.org/10.1038/s41467-020-18981-w}},
  doi          = {{10.1038/s41467-020-18981-w}},
  volume       = {{11}},
  year         = {{2020}},
}