Sowa, S. T., Galera-Prat, A., Wazir, S., Alanen, H. I., Maksimainen, M. M., & Lehtiö, L. (2021). A molecular toolbox for ADP-ribosyl binding proteins. Cell Reports Methods 1(8), 100121. https://doi.org/10.1016/j.crmeth.2021.100121
A molecular toolbox for ADP-ribosyl binding proteins
|Author:||Sowa, Sven T.1; Galera-Prat, Albert1; Wazir, Sarah1;|
1Faculty for Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, 90220 Oulu, Finland
|Online Access:||PDF Full Text (PDF, 3.3 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe2021121761679
|Publish Date:|| 2021-12-17
Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery.
Cell reports. Methods
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
1182 Biochemistry, cell and molecular biology
This work was funded by the Jane and Aatos Erkko and Sigrid Jusélius Foundations. The use of the facilities of the Biocenter Oulu Structural Biology core facility, member of Biocenter Finland, Instruct-ERIC Centre Finland, and FINStruct, as well as of Proteomics and Protein Analysis and Sequencing core facilities are gratefully acknowledged.
© 2021 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).