A ligand discovery toolbox for the WWE domain family of human E3 ligases

Münzker, Lena; Kimani, Serah W.; Fowkes, Milan M.; Dong, Aiping; Zheng, Hong; Li, Yanjun; Dasovich, Morgan; Zak, Krzysztof M.; Leung, Anthony K. L.; Elkins, Jonathan M.; Kessler, Dirk; Arrowsmith, Cheryl H.; Halabelian, Levon; Böttcher, Jark

doi:10.1038/s42003-024-06584-w

Title: A ligand discovery toolbox for the WWE domain family of human E3 ligases

Journal Article · Wed Jul 24 00:00:00 UTC 2024 · Communications Biology

DOI: https://doi.org/10.1038/s42003-024-06584-w · OSTI ID:2470235

Münzker, Lena ^[1];

^[2];

^[3]; Dong, Aiping ^[4]; Zheng, Hong ^[4];

^[4]; Dasovich, Morgan ^[5]; Zak, Krzysztof M. ^[1];

^[5]; Elkins, Jonathan M. ^[3];

^[1];

^[2];

^[4];

^[1]

Boehringer Ingelheim RCV GmbH & Co., Vienna (Austria)
Univ. of Toronto, ON (Canada); Princess Margaret Cancer Centre, Toronto, ON (Canada)
Univ. of Oxford (United Kingdom)
Univ. of Toronto, ON (Canada)
Johns Hopkins Univ., Baltimore, MD (United States)

The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, ¹⁵N-NMR-based binding affinity assays and ¹⁹F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2470235

Journal Information:: Communications Biology, Journal Name: Communications Biology Vol. 7; ISSN 2399-3642

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

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Title: A ligand discovery toolbox for the WWE domain family of human E3 ligases

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