Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Ciechanover, A., Orian, A. & Schwartz, A. L. Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 22, 442–51 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Békés, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat. Rev. Drug Discov. 21, 181–200 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–29 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Sakamoto, K. M. et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl Acad. Sci. USA 98, 8554–9 (2001).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Faust, T. B., Donovan, K. A., Yue, H., Chamberlain, P. P. & Fischer, E. S. Small-molecule approaches to targeted protein degradation. Annu. Rev. Cancer Biol. 5, 181–201 (2021).

    Article  Google Scholar 

  6. Ishida, T. & Ciulli, A. E3 ligase ligands for PROTACs: how they were found and how to discover new ones. SLAS Discov. 26, 484–502 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Harper, J. W. & Schulman, B. A. Cullin–RING ubiquitin ligase regulatory circuits: a quarter century beyond the F-box hypothesis. Annu. Rev. Biochem. 90, 1–27 (2021).

    Article  Google Scholar 

  8. Lin, H.-C. et al. C-terminal end-directed protein elimination by CRL2 ubiquitin ligases. Mol. Cell 70, 602–613 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Koren, I. et al. The eukaryotic proteome is shaped by E3 ubiquitin ligases targeting C-terminal degrons. Cell 173, 1622–1635 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Meszaros, B., Kumar, M., Gibson, T. J., Uyar, B. & Dosztanyi, Z. Degrons in cancer. Sci. Signal 10, eaak9982 (2017).

    Article  PubMed  Google Scholar 

  11. Rusnac, D. V. et al. Recognition of the diglycine C-end degron by CRL2KLHDC2 ubiquitin ligase. Mol. Cell 72, 813–822 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jevtić, P., Haakonsen, D. L. & Rapé, M. An E3 ligase guide to the galaxy of small-molecule-induced protein degradation. Cell Chem. Biol. 28, 1000–1013 (2021).

    Article  PubMed  Google Scholar 

  13. Bricelj, A., Steinebach, C., Kuchta, R., Gütschow, M. & Sosič, I. E3 ligase ligands in successful PROTACs: an overview of syntheses and linker attachment points. Front. Chem. 9, 707317 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kannt, A. & Đikić, I. Expanding the arsenal of E3 ubiquitin ligases for proximity-induced protein degradation. Cell Chem. Biol. 28, 1014–1031 (2021).

    Article  CAS  PubMed  Google Scholar 

  15. Buckley, D. L. et al. Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction. J. Am. Chem. Soc. 134, 4465–4468 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dobrodziej, J. et al. Evaluating ligands for ubiquitin ligases using affinity beads. Methods Mol. Biol. 2365, 59–75 (2021).

    Article  CAS  PubMed  Google Scholar 

  17. Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Riching, K. M. et al. Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action. ACS Chem. Biol. 13, 2758–2770 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Zou, Y., Rojas-Pierce, M., Raikhel, N. V. & Pirrung, M. C. Preparation of methyl ester precursors of biologically active agents. Biotechniques 44, 377–384 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Raina, K. et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl Acad. Sci. USA 113, 7124–7129 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ohba, K. Sulfonamide or sulfinamide compound having effect of inducing BRD4 protein degradation and pharmaceutical use thereof. World Intellectual Patent Organization WO/2021/157684 (2021).

  22. Li, Y.-D. et al. Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders. Preprint at biorXiv https://doi.org/10.1101/2023.02.14.528208 (2023).

  23. Hsia, O. et al. An intramolecular bivalent degrader glues an intrinsic BRD4–DCAF16 interaction. Preprint at biorXiv https://doi.org/10.1101/2023.02.14.528511 (2023).

  24. Pla‐Prats, C., Cavadini, S., Kempf, G. & Thomä, N. H. Recognition of the CCT5 di‐Glu degron by CRL4DCAF12 is dependent on TRiC assembly. EMBO J. 42, e112253 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Canzani, D., Rusnac, D.-V., Zheng, N. & Bush, M. F. Degronomics: mapping the interacting peptidome of a ubiquitin ligase using an integrative mass spectrometry strategy. Anal. Chem. 91, 12775–12783 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Punjani, A. & Fleet, D. J. 3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J. Struct. Biol. 213, 107702 (2021).

    Article  CAS  PubMed  Google Scholar 

  27. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  28. Nguyen, H. C., Yang, H., Fribourgh, J. L., Wolfe, L. S. & Xiong, Y. Insights into Cullin–RING E3 ubiquitin ligase recruitment: structure of the VHL–EloBC–Cul2 complex. Structure 23, 441–449 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chirnomas, D., Hornberger, K. R. & Crews, C. M. Protein degraders enter the clinic—a new approach to cancer therapy. Nat. Rev. Clin. Oncol. 20, 265–278 (2023).

    Article  CAS  PubMed  Google Scholar 

  30. Schapira, M., Calabrese, M. F., Bullock, A. N. & Crews, C. M. Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug Discov. 18, 949–963 (2019).

    Article  CAS  PubMed  Google Scholar 

  31. Lee, J. et al. Discovery of E3 ligase ligands for target protein degradation. Molecules 27, 6515 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Poirson, J. et al. Proteome-scale induced proximity screens reveal highly potent protein degraders and stabilizers. Preprint at biorXiv https://doi.org/10.1101/2022.08.15.503206 (2022)

  33. Röth, S. et al. Screening of E3 ligases uncovers KLHDC2 as an efficient proximity-induced degrader of K-RAS, STK33, β-catenin and FoxP3. SSRN Electron J. https://doi.org/10.2139/ssrn.4214930 (2022).

    Article  Google Scholar 

  34. Kim, Y. et al. Targeted kinase degradation via the KLHDC2 ubiquitin E3 ligase. Cell Chem Biol https://doi.org/10.1016/j.chembiol.2023.07.008 (2023)

  35. Sherpa, D., Chrustowicz, J. & Schulman, B. A. How the ends signal the end: regulation by E3 ubiquitin ligases recognizing protein termini. Mol. Cell. 82, 1424–1438 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Balaji, V. & Hoppe, T. Regulation of E3 ubiquitin ligases by homotypic and heterotypic assembly. F1000Res. 9, F1000 Faculty Rev-88 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Mallik, S. & Kundu, S. Topology and oligomerization of mono- and oligomeric proteins regulate their half-lives in the cell. Structure 26, 869–878 (2018).

    Article  CAS  PubMed  Google Scholar 

  38. Mohamed, W. I. et al. The CRL4DCAF1 Cullin–RING ubiquitin ligase is activated following a switch in oligomerization state. EMBO J. 40, e108008 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Reichermeier, K. M. et al. PIKES analysis reveals response to degraders and key regulatory mechanisms of the CRL4 network. Mol. Cell 77, 1092–1106 (2020).

    Article  CAS  PubMed  Google Scholar 

  40. Henneberg, L. T. et al. Activity-based profiling of cullin–RING ligase networks by conformation-specific probes. Nat Chem Biol https://doi.org/10.1038/s41589-023-01392-5 (2023)

  41. Scott, D. C. et al. E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity. Mol. Cell 83, 770–786 (2022).

    Article  Google Scholar 

  42. Cardote, T. A. F., Gadd, M. S. & Ciulli, A. Crystal structure of the Cul2–Rbx1–EloBC–VHL ubiquitin ligase complex. Structure 25, 901–911 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Buckley, D. L. et al. Targeting the von Hippel–Lindau E3 Ubiquitin Ligase Using Small Molecules To Disrupt the VHL/HIF-1α Interaction. J. Am. Chem. Soc. 134, 4465–4468 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schrödinger Release 2023-1: Maestro (Schrödinger, 2021).

  45. Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R. & Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 27, 221–234 (2013).

    Article  ADS  PubMed  Google Scholar 

  46. Bowers, K. J. et al. Scalable algorithms for molecular dynamics simulations on commodity clusters. in SC '06: Proc. of the 2006 ACM/IEEE Conference on Supercomputing 43 (IEEE, 2006)

  47. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2010).

    Article  ADS  Google Scholar 

  48. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  50. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  ADS  PubMed  Google Scholar 

  51. Bich, C. et al. Reactivity and applications of new amine reactive cross-linkers for mass spectrometric detection of protein−protein complexes. Anal. Chem. 82, 172–179 (2010).

    Article  CAS  PubMed  Google Scholar 

  52. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  53. Liebschner, D. et al. Macromolecular structure determination using X‐rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Biol. Crystallogr. 75, 861–877 (2019).

    Article  ADS  CAS  Google Scholar 

Download references

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

Authors
  1. Christopher M. Hickey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katherine M. Digianantonio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kurt Zimmermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alicia Harbin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Connor Quinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Avani Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peter Gareiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Amanda Chapman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bernadette Tiberi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jennifer Dobrodziej
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. John Corradi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Angela M. Cacace
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. David R. Langley
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Miklós Békés
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Miklós Békés.

Ethics declarations

Competing interests

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

Peer review

Peer review information

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.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

Supplementary information

Supplementary Methods

Supplementary chemistry methods for compounds

Reporting Summary

Source data

Source Data Figs. 1–4 and Extended Data Figs. 2–4

All graph data in excel as tabs per figure panel.

Source Data Figs. 2–4 and Extended Data Figs. 1, 2, and 4–7

All uncropped images as blots

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-023-01146-w

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research