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Co-opting the E3 ligase KLHDC2 for targeted protein degradation by small molecules

Nature Structural & Molecular Biology volume 31, pages 311–322 (2024)Cite this article

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Abstract

Targeted protein degradation (TPD) by PROTAC (proteolysis-targeting chimera) and molecular glue small molecules is an emerging therapeutic strategy. To expand the roster of E3 ligases that can be utilized for TPD, we describe the discovery and biochemical characterization of small-molecule ligands targeting the E3 ligase KLHDC2. Furthermore, we functionalize these KLHDC2-targeting ligands into KLHDC2-based BET-family and AR PROTAC degraders and demonstrate KLHDC2-dependent target-protein degradation. Additionally, we offer insight into the assembly of the KLHDC2 E3 ligase complex. Using biochemical binding studies, X-ray crystallography and cryo-EM, we show that the KLHDC2 E3 ligase assembles into a dynamic tetramer held together via its own C terminus, and that this assembly can be modulated by substrate and ligand engagement.

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Fig. 1: Discovery of high-affinity small-molecule ligands, by structure-based de novo design, targeting the substrate-binding kelch domain of KLHDC2.
Fig. 2: Functionalized KLHDC2-targeting small molecules, when incorporated into heterobifunctional molecules, bring KLHDC2 and target proteins into closer proximity.
Fig. 3: Degradation of BET-family proteins and AR by KLHDC2-based PROTACs.
Fig. 4: Purified full-length KLHDC2–EloB–EloC E3 ligase complex is a dynamic oligomer held together in part by its own C termini.
Fig. 5: Cryo-EM structure at 3.8 Å of the full-length, apo-KLHDC2–EloB–EloC complex reveals a tetramer.

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Data availability

Data are available and coordinates have been deposited at the PDB under accession numbers PDB 8SGE (KLHDC2KD–KDRLKZ-1), PDB 8SGF (KLHDC2KD–peptide C terminus), and PDB 8SH2 (apo-KBC) for the crystal structures. Cryo-EM maps for KLHDC2 have also been deposited at the Electron Microscopy Data Bank under accession numbers EMD-40477 (apo-KBC). In addition, the following published crystallographic data sets were used in this study: PDB 6DO3; PDB 5T35. Source data are provided with this paper.

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Acknowledgements

We thank I. Taylor for helpful comments on the manuscript; I. Drulyte, Y. Xiong, and A. Fathizadeh for helpful tips and discussions on cryo-EM data processing; and C.-j. Lee and H. Huang for assistance with crystallography data.

Author information

Author notes

  1. Bernadette Tiberi

    Present address: Genetics, Genomics and Cancer Biology Graduate Program, Thomas Jefferson University, Philadelphia, PA, USA

  2. Jennifer Dobrodziej

    Present address: Accent Therapeutics Inc, Lexington, MA, USA

  3. These authors contributed equally: Christopher M. Hickey, Katherine M. Digianantonio.

Authors and Affiliations

  1. Arvinas, Inc, New Haven, CT, USA

    Christopher M. Hickey, Katherine M. Digianantonio, Kurt Zimmermann, Alicia Harbin, Connor Quinn, Avani Patel, Peter Gareiss, Amanda Chapman, Bernadette Tiberi, Jennifer Dobrodziej, John Corradi, Angela M. Cacace, David R. Langley & Miklós Békés

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Contributions

Data collection and analysis—C.M.H., K.M.D., A.H., K.Z., D.R.L., A. Chapman, A.P., C.Q., P.G., B.T., J.D., J.C., M.B.; manuscript preparation—C.M.H., K.M.D., A.H., K.Z., D.R.L., A.M.C., M.B. (writing, reviewing, figure preparation). All authors have read and commented on the manuscript. Corresponding author: M.B.

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Correspondence to Miklós Békés.

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All authors are, or have been, employees and shareholders of Arvinas Inc, which is developing PROTAC molecules for therapeutic applications.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Dimitris Typas was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Additional biochemical characterization of KLHDC2 ligands.

a) In vitro ubiquitylation reactions of a Cy5-conjugated USP1 C-terminal peptides by KLHDC2/EloB/EloC (representative gel shown of two independent experiments). b) In vitro ubiquitylation reactions of a Cy5-conjugated USP1 C-terminal peptides in the presence and absence of KLHDC2-targeting small molecules (representative gel shown of two independent experiments). c) Structure of a biotin-conjugated KLHDC2 ligand used in Fig. 2a,b. d) KLHDC2 pull-down using probe above in C) in the absence and presence of competing excess KDRLKZ-1, analyzed by anti-KLHDC2 immunoblot, performed in duplicate. e) Ternary complex SPR sensograms and kinetic values (related to Figs. 2d and e), K2–B4–3 sensograms are duplicated in this Extended Data panel for completion).

Source data

Extended Data Fig. 2 Additional cell-based characterization of KLHDC2-based degraders.

a) HiBiT-BRD4 levels were measured following 24 hr treatment of PC-3_HiBiT-BRD4 cells with the indicated PROTACs (n = 4 replicates). b) Endogenous BRD4 levels were measured across cell lines by immunoblots following 24 hr treatment with the K2-B4-3e PROTAC, and its controls (dotted lines included for clarity, one of two independent gels shown). c) Endogenous BRD4 levels were measured by immunoblot following 24 hr treatment of PC-3 cells with the K2-B4-3e PROTAC, with and without co-treatment with 1 mM MLN4924. d) Endogenous BRD4 and BRD3 levels were measured by immunoblots following 24 hr treatment of mouse A20 cells by the indicated PROTACs at 1 uM (n = 3 technical replicates). e) Left panel, HiBiT-BRD4 levels were measured from cells treated with the indicated siRNAs for 48 hours, then treated with the K2-B4-5e PROTAC for 4 hr (n = 2 biological replicates with n = 3 technical replicates); and right panel, anti-BRD4 immunoblot data from the same experiment (representative of triplicate blots). f) In a parallel set of wells, relative mRNA levels were measured by qPCR after siRNA treatment of cells, in triplicate (see Methods).

Source data

Extended Data Fig. 3 UPS-wide siRNA screen against compound 1.

a) Structure of compound 1 (left) and (right) PC-3_HiBiT-BRD4 cells were treated in triplicate for a total of 72 hours with siRNA to the indicted gene (or no siRNA as a control) with either DMSO of 50 nM compound 1 (in triplicate) added for the final 2 hours before collecting samples for immunoblots for BRD2. b) PC-3_HiBiT-BRD4 cells were treated with the indicated potential inhibitors of compound 1 action 30 min prior to treatment with the indicated concentrations of compound 1 for 2 hours. NAEi was used at 1 μM while JQ1 and E7820 were used at 10 μM. The ‘DMSO’ curve in this part shows mean −/+ SD data based on duplicate measures per dose whereas the other curves are based on singlicate measures per dose. c) PC-3_HiBiT-BRD4 cells were screened with a siRNA library covering 968 genes, listed on the x-axis. Percent of the mean for the ‘no degrader wells’ (from a total of 32 wells on two control plates) is graphed for the 6 individual data points per gene (from 6 wells, duplicates for 3 separate siRNAs per gene). The green horizontal line shows the threshold used for calling hits, based on mean + 3-fold SD of the ‘no siRNA’ condition across plates. The cluster showing many points with relatively high values for gene #s 140-175 are all genes for proteasome subunits. The genes for ubiquitin, also common hits in the screen, are at positions 228–230 on the graph. d) Table showing siRNA screen data for genes with a hit score of 3 out of 6 or greater, not including genes for ubiquitin or the proteasome. The ‘no siRNA’ wells in the screen (n = 268, 4 per 96-well plate) had a mean of 7.4% with a SD of 0.7%. We used a cutoff of >9.5%, which is 7.4% + 3xSD, to call a well a hit. Hit score refers to how many wells for a given gene were called hits. ‘Mean % no degrader’ in this table includes all 6 values for the gene, even those wells which were not hits.

Source data

Extended Data Fig. 4 Characterization of the NLD (NanoLuc-degron) system for KLHDC2.

a) Cartoon representation of the NLD assay. b) MLN4924 dose-response curve showing the stabilization of a CRL2KLHDC2-degron in the NLD assay using the Nano-Glo detection system (n = 2 replicates shown). c) CRL2KLHDC2-degron NLD signal is stabilized with KLHDC2 siRNA treatments shown by a Nano-Glo readout (n = 3 replicates), as well as by anti-HA immunoblot for the stabilized protein. d) Nano-Glo readout of a CRL2KLHDC2-degron and a CRL2KLHDC3-degron stability by KLHDC2, KLHDC3 and KLHDC10 siRNAs – showing specific stabilization by the respective degrons for KLHDC2 and KLHDC3. Error bars represent mean + SD.

Source data

Extended Data Fig. 5 Additional characterization of KBC complexes by MALDI and aSEC.

a) Peaks in the high mass MALDI experiment show that KLHDC2/EloB/EloC is a higher-order oligomer that dissociates into smaller subcomplexes upon binding substrate. b) SDS-PAGE gels of KBC complexes incubated with peptides shown in Fig. 4e, stained with Coomassie.

Source data

Extended Data Fig. 6 Cryo-EM processing workflow of the KLHDC2-EloB-EloC tetramer.

a) SDS-PAGE analysis of cross-linked KBC complexes in preparation for cryoEM. b) Overview of processing workflow, from raw micrographs to final map shown in Fig. 5a. Upon ab initio calculation, multiple 3D classes were obtained that demonstrate dimeric, trimeric, and tetrameric assemblies of KBC. Further analysis was focused on the tetrameric assembly. All steps performed in cryoSPARC 3.3.1 and 4.1.1. c) Local resolution mapped onto final map. d) FSC plot. e) Viewing distribution for the final reconstruction.

Source data

Extended Data Fig. 7 Characterization of KBC complexes by CUL2 binding and compounds.

a) Overlay of the structure models of the two non-identical KBC units within the tetramer. b) EloC from PDB ID 5n4w aligned to model in paper. Orange KLHDC2 is not competent for binding Cul2 (left). Purple KLHDC2 is competent for binding Cul2 (right). c) Size Exclusion chromatograms demonstrating elution profiles of 10 µM KBC (grey), 10 µM Cul2/Rbx1 (orange), and a mixture of 10 µM KBC and 10 µM Cul2/Rbx1 (blue). The elution of the mixture to earlier elution volumes demonstrates interaction of KBC and Cul2/Rbx1, which can also be seen in the SDS-PAGE gel of the fractions collected (stained with Coomassie). d) SDS-PAGE gels of KBC complexes incubated with small molecules shown in Fig. 5f.

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Hickey, C.M., Digianantonio, K.M., Zimmermann, K. et al. Co-opting the E3 ligase KLHDC2 for targeted protein degradation by small molecules. Nat Struct Mol Biol 31, 311–322 (2024). https://doi.org/10.1038/s41594-023-01146-w

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