A molecular toolbox for ADP-ribosyl binding proteins
Sowa, Sven T.; Galera-Prat, Albert; Wazir, Sarah; Alanen, Heli I.; Maksimainen, Mirko M.; Lehtiö, Lari (2021-11-11)
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
© 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/).
https://creativecommons.org/licenses/by-nc-nd/4.0/
https://urn.fi/URN:NBN:fi-fe2021121761679
Tiivistelmä
Summary
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.
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