A Ligand Discovery Toolbox for the WWE Domain Family of Human E3 Ligases

Overview

Journal Commun Biol

Specialty Biology

Date 2024 Jul 24

PMID 39048679

Authors

Lena Munzker

Serah W Kimani

Milan M Fowkes

Aiping Dong

Hong Zheng

Yanjun Li

Morgan Dasovich

Krzysztof M Zak

Anthony K L Leung

Jonathan M Elkins

Dirk Kessler

Cheryl H Arrowsmith

Levon Halabelian

Jark Bottcher

Affiliations

Soon will be listed here.

Abstract

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, N-NMR-based binding affinity assays and F-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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