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Dynamic molecular architecture and substrate recruitment of cullin3–RING E3 ligase CRL3KBTBD2

Nature Structural & Molecular Biology volume 31, pages 336–350 (2024)Cite this article

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Abstract

Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3–RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2–p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.

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Fig. 1: Cryo-EM structures of CRL3KBTBD2 assembly.
Fig. 2: Interface analysis of CRL3KBTBD2 assembly.
Fig. 3: Cryo-EM structure of the CRL3KBTBD2–p85α.
Fig. 4: Structure of the CRL3KBTBD2~N8 and CRL3KBTBD2~N8–CSNH138A.
Fig. 5: Structure of CRL3KBTBD2~N8–CSNH138A and interface analysis.
Fig. 6: CRL3KBTBD2 catalytic cycle.

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Data availability

Coordinates and maps associated with data reported in this manuscript were deposited to the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) with accession numbers EMD-34199 and PDB 8GQ6 (CRL3KBTBD2 dimer), EMD-34451 and PDB 8H35 (CRL3KBTBD2 octamer), EMD-34450 and PDB 8H34 (CRL3KBTBD2 hexamer), EMD-34449 and PDB 8H33 (CRL3KBTBD2 tetramer), EMD-34453 and PDB 8H37 (CRL3KBTBD2–p85α tetramer), EMD-34452 and PDB 8H36 (CRL3KBTBD2–p85α dimer), EMD-34454 (CRL3KBTBD2–p85α local), EMD-34474 and PDB 8H3R (CRL3KBTBD2~N8), EMD-34467 and PDB 8H3F (CRL3KBTBD2–CSN), EMD-34466 (CRL3KBTBD2–CSNΔ3/8), EMD-34462 and PDB 8H3A (CRL3KBTBD2~N8Δ–CSN), EMD-34463 (CRL3KBTBD2~N8Δ–CSNΔ3/8), EMD-34455 and PDB 8H38 (CRL3KBTBD2~N8–CSNH138A), EMD-34457 (CRL3KBTBD2~N8–CSNH138A,CSN1/3/7/8 local), EMD-34458 (CRL3KBTBD2~N8–CSNH138A, N8/CSN5/6 local), EMD-34456 (CRL3KBTBD2~N8–CSNH138A, CUL3CTD~N8–CSN1/2/4/5/6 local), EMD-34459 (CRL3KBTBD2~N8–CSNH138A, composite map after local refinement), EMD-34460 (CRL3KBTBD2~N8–CSNH138A, Δ3/8), EMD-34461(CRL3KBTBD2~N8–CSNH138A, 2:2), EMD-34473 and PDB 8H3Q (CUL3–RBX1–CAND1). Source data are provided with this paper.

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Acknowledgements

We thank Center of Cryo-Electron Microscopy, Fudan University for assistance with cryo-EM data collection. We thank X. Yu Shanghai at Institute of Materia Medica, Chinese Academy of Sciences for assistance with cryo-EM grids screening. This work was supported by grants from the National Natural Science Foundation of China (grant no. 81900729 to L.S.), the Science and Technology Commission of Shanghai Municipality (grant no. 23ZR1413700 to L.S.), the US National Institutes of Health (grant nos. K99 DK115766 and R00 DK115766 to Z.Z.; R01 AI125581 and U19 AI100627 to B.B.) and the Lyda Hill Foundation (B.B.).

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  1. These authors contributed equally: Yuxia Hu, Zhao Zhang, Qiyu Mao, Xiang Zhang, Aihua Hao.

Authors and Affiliations

  1. Shanghai Fifth People’s Hospital, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Key Laboratory of Medical Epigenetics and Institutes of Biomedical Sciences, Fudan University, Shanghai, China

    Yuxia Hu, Qiyu Mao, Xiang Zhang, Aihua Hao, Yeda Wang, Lin Han, Wuqiang Zhan, Qianying Liu, Zhenguo Chen & Lei Sun

  2. Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhao Zhang, Yu Xun, Eva Marie Y. Moresco & Bruce Beutler

  3. Division of Endocrinology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhao Zhang & Yu Xun

  4. National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Science, Shanghai, China

    Yue Yin & Chao Peng

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L.S. and B.B. initiated, planned and supervised the project. Y.H. performed the protein expression and purification with assistance from Y.W., A.H., W.Z., L.H. and Q.L. Z.C., X.Z. and Q.M. collected the cryo-EM data and solved the structures with assistance from Y.H. Q.M. and L.S. analyzed the structures. Y.Y. and C.P. performed the XL-MS assay. Z.Z. and Y.X. performed the interaction assays of KBTBD2 and p85α mutations. The manuscript was written by Y.H., L.S., Z.Z., E.M.Y.M. and B.B., and was approved by all the authors.

Corresponding authors

Correspondence to Zhenguo Chen, Bruce Beutler or Lei Sun.

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Competing interests

The authors declare no competing interests.

Peer review

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Nature Structural & Molecular Biology thanks Gilbert Privé and the other, anonymous, reviewer(s) 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. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Size exclusion chromatography and negative stain micrographs of KBTBD2, p85α, CRL3KBTBD2, CRL3KBTBD2–p85α and CRL3KBTBD2~N8.

a–e, Size exclusion chromatography and negative stain micrographs of KBTBD2 (a), p85α (b), CRL3KBTBD2 (c), CRL3KBTBD2–p85α (d) and CRL3KBTBD2~N8 (e). Left: SEC profiles, the chromatographic separation of the standard proteins is shown as a grey line and the theoretical molecular weights are shown above. Middle: Coomassie−stained SDS-PAGE analysis of the peak fraction of SEC. Right: representative micrographs. The purification was repeated independently more than 3 times with similar results.

Source data

Extended Data Fig. 2 Size exclusion chromatography and negative stain micrographs of CSN in complex with neddylated and non-neddylated CRL3KBTBD2.

a, CRL3KBTBD2. b, CSN. c, CSNH138A. d, CRL3KBTBD2–CSN. e, CRL3KBTBD2~N8Δ–CSN. f, CRL3KBTBD2~N8–CSNH138A. Left: SEC profiles, the chromatographic separation of the standard proteins is shown as a grey line and the theoretical molecular weights are shown above. Middle: Coomassie−stained SDS-PAGE analysis of the peak fraction of SEC. Right: representative micrographs. The purification was repeated independently more than 3 times with similar results.

Source data

Extended Data Fig. 3 Structure of the CRL3KBTBD2 monomer.

a, Ribbon representation of CRL3KBTBD2 monomer with subdomains colored differently. b, The cryo-EM map and the model of the CUL3CTD and RBX1, showing that the N-terminal β-strand of RBX1 (RBX1NTD) inserts into the CUL3CTD, forming a stable β-sheet. The subdomains of the CUL3CTD are colored individually. Three zinc ions in RBX1 are shown as green spheres. c, Analysis of interface between KBTBD2 and CUL3. The residues located at the contact surface are shown as sticks. d, Ribbon representation of the KBTBD2Kelch domain in side and top views. The Kelch domain contains six kelch-repeats and each consists of four β-strands, packing into a helix cluster structure. e, Superposition of five Kelch domains: KLHL12 (PDB: 2VPJ), KLHL2 (PDB: 2XN4), KBTBD10 (PDB: 2WOZ), KEAP1 (PDB: 2FLU), KBTBD2 (this study). f-g, Sequence and structural alignment of BTB domain of KBTBD2 with another four BTBs.

Extended Data Fig. 4 Size-exclusion chromatography and negative staining EM micrographs of CRL3KBTBD2 mutants.

a, Size-exclusion chromatography (SEC) comparison of CRL3KBTBD2 mutants on H24 (Q489A, H490A, Q492A, T494A, V496A), H25 (R537A, K542A, S544A), WHB (S713A, R714A, K715A, K716A). The chromatographic separation of the standard proteins is shown as a grey line and the theoretical molecular weights are shown above. b-f, Coomassie-stained SDS-PAGE gel (Left) and negative-stain EM analysis (Right) of the CRL3KBTBD2_WT (b), CRL3KBTBD2_H24 (c), CRL3KBTBD2_H25 (d), CRL3KBTBD2_WHB (e), CRL3KBTBD2_H2425 (f), elution fractions. The purification was repeated independently more than 3 times with similar results.

Source data

Extended Data Fig. 5 Chemical cross-linking mass spectrometry (XL-MS) analysis of the KBTBD2–p85α complex.

a, The circular plot of cross-links identified for the KBTBD2-p85α complex. b, KBTBD2–p85α inter- (blue) and intra-subunit (orange) cross-links projected onto the cryo-EM structure of CRL3KBTBD2–p85α. c, Euclidean distances for cross-links shown in (a). d-e, Cryo-EM density maps of CRL3KBTBD2–p85α dimer fitted with the corresponding models of KBTBD2 and p85α. The domains are labeled in different colors.

Extended Data Fig. 6 Structure comparison of CRL3KBTBD2 and CRL3KBTBD2–p85α.

a, b, Side view and bottom view of CRL3KBTBD2 octamer (a) and hexamer (b). c, d, Side view and bottom view of CRL3KBTBD2 tetramer (c) and CRL3KBTBD2–p85α tetramer (d), showing that CRL3KBTBD2 tetramer contains two dimers forming an angle of ~90°, while CRL3KBTBD2–p85α tetramer contains two dimers forming an angle of ~180°. e, Fitting CRL3KBTBD2–p85α dimer models into CRL3KBTBD2 octamer maps, showing that p85α clashes with the neighboring KBTBD2s. f, Structural comparison of CRL3KBTBD2 octamer (gray) with two neighbor CRL3KBTBD2–p85α dimers (colored), the clashes area is indicated by circle. g, The details of clashes between KBTBD2 (gray) and p85α (colored).

Extended Data Fig. 7 Structure analysis of CRL3KBTBD2~N8 structure.

a, Cryo-EM density map and models of the CUL3CTD and RBX1 in CRL3KBTBD2~N8 structure. The subdomains of CUL3CTD are colored individually. The neddylation site Lys712 is shown as green spheres. b, Neddylation assay of CRL3KBTBD2. n, the number of biological independent replicates. c-e, Structural comparison of CRL3KBTBD2~N8 with other neddylatd CRLs: CUL1~N8 (PDB: 6TTU) and CUL5 ~N8 (PDB: 3DQV). The N8 sides are shown as atoms in green. f-h, Fitting CRL3KBTBD2~N8 into CRL3KBTBD2 multimer maps indicates structural clashes (circled) between RBX1 and CUL34HB subdomain from the neighboring CUL3CTD.

Extended Data Fig. 8 Cryo-EM maps of CSN in complex with neddylated or non-neddylated CRL3KBTBD2.

a, Overall Cryo-EM maps of CRL3KBTBD2–CSN containing one whole CSN molecules, and (b) one CSN missing the CSN3/8 density. c, Overall Cryo-EM maps of CRL3Kbtbd2~N8Δ–CSN containing one whole CSN molecules, and (d) one CSN missing the CSN3/8 density. e, Overall Cryo-EM maps of CRL3KBTBD2~N8–CSNH138A containing two CSN molecules (one with the whole CSN and another missing the CSN3/8 density), (f) CRL3KBTBD2~N8–CSNH138A containing one whole CSN and missing the CUL3/RBX1 subunits, and (g) CRL3KBTBD2~N8–CSNH138A containing one CSN missing CSN3/8 subunits and weak density in CUL3/RBX1 subunits. h, Summary of the conformation distribution of each state and the resolution of each structure.

Extended Data Fig. 9 Structural comparison of CSN in complex with neddylated or non-neddylated CRL3KBTBD2.

a, Structural comparison of CSN in apo CSN (gray) and CRL3KBTBD2~N8–CSNH138A (colored). b, Comparison of CSN in apo CSN (gray) and CRL3KBTBD2–CSN (colored). c, Comparison of CSN in apo CSN (gray) and CRL3KBTBD2~N8Δ–CSN (colored). d, Comparison of CSN in CRL3KBTBD2~N8Δ–CSN (gray) and CRL3KBTBD2~N8–CSNH138A (colored). e, Comparison of CSN in CRL3KBTBD2–CSN (gray) and CRL3KBTBD2~N8–CSNH138A (colored). f-i, The CSN5/6 heterodimers in apo CSN (f), CRL3KBTBD2~N8–CSNH138A ‘deactivating’ state (g), CRL3KBTBD2~N8Δ–CSN ‘deactivated’ state (h) and CRL3KBTBD2–CSN ‘inhibited’ state (i) are shown in cartoon representation. j, Alignment of RBX1 between different states of CRL3KBTBD2. k, Alignment of CUL3WHB and CUL3WHA subdomains between different states of CRL3KBTBD2. The CUL3WHB structures of different states were displayed at right.

Extended Data Fig. 10 Structure of the CUL3–RBX1–CAND1 complex.

a, The schematic representation of the domain composition of CAND1. b, Cryo-EM density of CAND1 and ribbon model of the CUL3–RBX1–CAND1 complex. The neddylation site K712 in WHB subdomain is shown in green spheres. c, d, Superimposing KBTBD2BTB-BACK/CUL3NTD structure with CUL3–RBX1–CAND1, showing that CAND1 clashes with BTB domain. The KBTBD2BTB-BACK is shown in gray surface. e, the interface between CAND1 N-terminal arch and CUL3WHB. f, Superimposition of the CUL3–RBX1–CAND1 model (multi-colored) with CUL1–RBX1–CAND1 (PDB: 1U6G, gray) and CUL4B–RBX1–CAND1 (PDB: 4AOC, blue).

Supplementary information

Supplementary Information

Supplementary Figs. 1–16 and Tables 1–3 and captions for Videos 1–5.

Reporting Summary

Peer Review File

Supplementary Video 1

Structural assembly of CRL3KBTBD2 dimer, tetramer, hexamer and octamer.

Supplementary Video 2

Substrate recruitment of CRL3KBTBD2, showing the structure transition from CRL3KBTBD2 to CRL3KBTBD2–p85α.

Supplementary Video 3

Activation of CRL3KBTBD2 by NEDD8, showing the structure transition from CRL3KBTBD2 to CRL3KBTBD2~N8.

Supplementary Video 4

Structure transition from activated state (CRL3KBTBD2~N8) to deactivating state CRL3KBTBD2~N8–CSNH138A.

Supplementary Video 5

Structure transition from deactivating state CRL3KBTBD2~N8–CSNH138A to deactivated state CRL3KBTBD2~N8Δ–CSN.

Source data

Source Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 1

Original SDS–PAGE.

Source Data Extended Data Fig. 2

Original SDS–PAGE.

Source Data Extended Data Fig. 4

Original SDS–PAGE.

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Hu, Y., Zhang, Z., Mao, Q. et al. Dynamic molecular architecture and substrate recruitment of cullin3–RING E3 ligase CRL3KBTBD2. Nat Struct Mol Biol 31, 336–350 (2024). https://doi.org/10.1038/s41594-023-01182-6

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