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

Author

Listed:
  • Kathrin Nowak

    (University of Zurich
    University of Zurich)

  • Florian Rosenthal

    (University of Zurich)

  • Tobias Karlberg

    (Karolinska Institute)

  • Mareike Bütepage

    (RWTH Aachen University)

  • Ann-Gerd Thorsell

    (Karolinska Institute)

  • Birgit Dreier

    (University of Zurich)

  • Jonas Grossmann

    (Functional Genomics Center Zurich, ETH Zurich and University of Zurich
    SIB Swiss Institute of Bioinformatics, Quartier Sorge - Batiment Amphipole)

  • Jens Sobek

    (Functional Genomics Center Zurich, ETH Zurich and University of Zurich)

  • Ralph Imhof

    (University of Zurich)

  • Bernhard Lüscher

    (RWTH Aachen University)

  • Herwig Schüler

    (Karolinska Institute)

  • Andreas Plückthun

    (University of Zurich)

  • Deena M. Leslie Pedrioli

    (University of Zurich)

  • Michael O. Hottiger

    (University of Zurich)

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.

Suggested Citation

  • Kathrin Nowak & Florian Rosenthal & Tobias Karlberg & Mareike Bütepage & Ann-Gerd Thorsell & Birgit Dreier & Jonas Grossmann & Jens Sobek & Ralph Imhof & Bernhard Lüscher & Herwig Schüler & Andreas Pl, 2020. "Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins," Nature Communications, Nature, vol. 11(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-18981-w
    DOI: 10.1038/s41467-020-18981-w
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    Cited by:

    1. Ivo A. Hendriks & Sara C. Buch-Larsen & Evgeniia Prokhorova & Jonas D. Elsborg & Alexandra K.L.F.S. Rebak & Kang Zhu & Dragana Ahel & Claudia Lukas & Ivan Ahel & Michael L. Nielsen, 2021. "The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome," Nature Communications, Nature, vol. 12(1), pages 1-16, December.

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