Structure and dynamics of a pentameric KCTD5/CUL3/Gßγ E3 ubiquitin ligase complex.

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gßγ and reduces Gßγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gß1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gßγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gßγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gß and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gßγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

Development of Peptide Displacement Assays to Screen for Antagonists of DDB1 Interactions.

The DNA damage binding protein 1 (DDB1) is an essential component of protein complexes involved in DNA damage repair and the ubiquitin-proteasome system (UPS) for protein degradation. As an adaptor protein specific to Cullin-RING E3 ligases, DDB1 binds different receptors that poise protein substrates for ubiquitination and subsequent degradation by the 26S proteasome. Examples of DDB1-binding protein receptors are Cereblon (CRBN) and the WD-repeat containing DDB1- and CUL4-associated factors (DCAFs). Cognate substrates of CRBN and DCAFs are involved in cancer-related cellular processes or are mimicked by viruses to reprogram E3 ligases for the ubiquitination of antiviral host factors. Thus, disrupting interactions of DDB1 with receptor proteins might be an effective strategy for anticancer and antiviral drug discovery. Here, we developed fluorescence polarization (FP)-based peptide displacement assays that utilize full-length DDB1 and fluorescein isothiocyanate (FITC)-labeled peptide probes derived from the specific binding motifs of DDB1 interactors. A general FP-based assay condition applicable to diverse peptide probes was determined and optimized. Mutagenesis and biophysical analyses were then employed to identify the most suitable peptide probe. The FITC-DCAF15 L49A peptide binds DDB1 with a dissociation constant of 68 nM and can be displaced competitively by unlabeled peptides at sub-µM to low nM concentrations. These peptide displacement assays can be used to screen small molecule libraries to identify novel modulators that could specifically antagonize DDB1 interactions toward development of antiviral and cancer therapeutics.

Discovery of Nedd4 auto-ubiquitination inhibitors.

E3 ubiquitin ligases are critical to the protein degradation pathway by catalyzing the final step in protein ubiquitination by mediating ubiquitin transfer from E2 enzymes to target proteins. Nedd4 is a HECT domain-containing E3 ubiquitin ligase with a wide range of protein targets, the dysregulation of which has been implicated in myriad pathologies, including cancer and Parkinson's disease. Towards the discovery of compounds disrupting the auto-ubiquitination activity of Nedd4, we developed and optimized a TR-FRET assay for high-throughput screening. Through selective screening of a library of potentially covalent compounds, compounds 25 and 81 demonstrated apparent IC50 values of 52 µM and 31 µM, respectively. Tandem mass spectrometry (MS/MS) analysis confirmed that 25 and 81 were covalently bound to Nedd4 cysteine residues (Cys182 and Cys867). In addition, 81 also adducted to Cys627. Auto-ubiquitination assays of Nedd4 mutants featuring alanine substitutions for each of these cysteines suggested that the mode of inhibition of these compounds occurs through blocking the catalytic Cys867. The discovery of these inhibitors could enable the development of therapeutics for various diseases caused by Nedd4 E3 ligase dysregulation.

The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of the transcription factor ERG.

The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.