Cullin–RING ubiquitin E3 ligase regulation by the COP9 signalosome

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Abstract

The cullin–RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A–RBX1–DDB1–DDB2 complex (CRL4ADDB2) monitors the genome for ultraviolet-light-induced DNA damage. CRL4ADBB2 is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4ADDB2 and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

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Figure 1: Cryo-EM structures of CSN–N8CRL4ADDB2 and CSN–N8CRL4A.
Figure 2: Conserved patches hold CSN and CRL4A together.
Figure 3: Structural changes in CSN induced by N8CRL4A binding.
Figure 4: A steric mechanism governs CRL4A binding to CSN.
Figure 5: Shared mechanisms of binding and regulation across CSN–CRLs complexes.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The cryo-EM structures are available from the Electron Microscopy Data Bank under accessions EMD-3313 (CSN–N8CRL4A at 6.7 Å), EMD-3314 (CSN–N8CRL4A at 6.4 Å), EMD-3315 (CSN–N8CRL4A at 8.8 Å) and EMD-3316 (CSN–N8CRL4ADDB2 at 8.3 Å). The negative-stain CSN–N8CRL3∆SPOP map is available under accession EMD-3317. The coordinates and structure factors for the CSN P1 crystal form have been deposited in the PDB under accession number 4WSN.

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Acknowledgements

We thank K. Böhm for technical assistance in protein expression and purification, D. Hess and J. Seebacher for mass spectrometry analysis, and M. Jones, B. Martoglio, R. Assenberg, C. Logel, I. Bechtold and M. Renatus for help developing the TR-FRET and PT22 CSN assays. We thank B. Anderson and A. Fecteau-Lefebvre (‘C-CINA’) for technical support and TEM maintenance. We are grateful to T. Walz and D. Barford for help and discussions. Part of this work was performed at beamline X10SA of the Swiss Light Source. This work was supported by the Novartis Research Foundation and grants to N.H.T. from the European Research Council (ERC-2014-ADG 666068 CSNCRL) and to H.S. from the Swiss initiative for Systems Biology (SystemsX.ch grant ‘C-CINA’). G.P. was supported by long-term fellowships of the European Molecular Biology Organization (EMBO; ALTF-1350-2013) and the Human Frontier Science Program (HFSP; LT000210/2014). W.I.M. was supported by the Boehringer Ingelheim Fonds.

Author information

S.C., E.S.F. and N.H.T, co-led the study. S.C. prepared the specimens for EM data collection and collected EM data with contributions from K.N.G.. S.C. performed EM data processing. E.S.F. developed the binding and activity assays with input from U.H., G.P. and A.P. G.M.L. designed and created CSN mutants with input from S.C. E.S.F, M.F., G.M.L. and W.A. purified proteins for crystallography, cryo-EM and biochemical assays. A.P. and E.S.F. performed functional assays and analysed the results. G.M.L. crystallized CSN. R.D.B. and G.M.L collected the X-ray diffraction data. R.D.B carried out the crystallographic analysis, built the cryo-EM models with input from S.C., and interpreted the results with S.C. and N.H.T.. K.N.G., R.S.P. and H.S. provided access to TEM microscopes and initial training. W.I.M. designed the RBX1–CSN4 fusion construct and purified protein for EM studies. R.E.J.B. and R.B.T. designed and performed the chemical syntheses. S.M. and K.S. performed cellular assays. S.C., E.S.F., R.D.B. and N.H.T. wrote the manuscript with input from G.P.

Correspondence to Nicolas H. Thomä.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Atomic models of the canonical CRL families.

a, CUL1–RBX1–SKP1–SKP2 (PDB accession codes 1LDK and 1FQV). b, CUL1–RBX1–SKP1–FBW7 (PDBs accession codes 1LDK and 2OVP). c, CUL2–RBX1–ElonginB–ElonginC–VHL (PDB accession codes 1LDK and 1VCB). d, Dimeric CUL3–RBX1–SPOP. The SPOP protein includes a BTB and a MATH domain. (PDB accession codes 1LDK, 4EOZ and 3HU6). e, CUL4A–RBX1–DDB1–DDB2 (PDB accession code 4A0K). f, CUL4A–RBX1–DDB1–CRBN (PDB accession codes 4A0K and 4CI1). g, CUL4A–RBX1–DDB1–CSA (PDB accession codes 4A0K and 4A11). h, CUL5–RBX1–ElonginB–ElonginC–SOCS2 (PDB accession codes 1LDK, 4JGH and 2C9W).

Extended Data Figure 2 Classification and refinement procedures for the CSN–N8CRL4ADDB2 complex.

a, Representative micrographs (scale bar, 50 nm; the whole data set includes 1,427 micrographs) and reference-free two-dimensional (2D) class averages (scale bar, 10 nm). b, After reference-free 2D classification, the initial model obtained from preliminary cryo-EM studies of CSN–N8CRL4ADDB2 was low-pass filtered to 60 Å and subjected to 3D classification to discard large misassembled particles. Class I was subjected to a second round of 2D classification and particles showing an intact assembly were merged with the particles included in the Class III, leading to 16,090 particles. The final model was refined to 8.3 Å resolution. c, Angular distribution plot. d, Gold-standard Fourier shell correlation curve (FSC).

Extended Data Figure 3 Classification and refinement procedures for the CSN–N8CRL4A complex.

a, Representative micrographs (scale bar, 50 nm; the whole data set includes 2,626 micrographs) and reference-free 2D class averages (scale bar, 10 nm). b, After 2D classification, the initial model obtained from preliminary cryo-EM studies of CSN–N8CRL4ADDB2 was low-pass filtered to 60 Å and subjected to a first round of 3D classification to discard large misassembled particles. Particles included in class I and class III were combined and refined in a 8.8 Å reconstruction map. To improve the resolution, we performed further 3D classification into two classes. The most populated (73%, 63,269 particles) class shows better resolution and finer details as suggested by the slice-through Z of the map (black circles with with particles on the left and right side of the 3D model). Refinement with a soft mask around the more rigid part of the complex led to a 6.4 Å resolution map. Refinement of the same set of particles with a soft mask around the whole complex let to a 6.7 Å resolution map. The 6.7 Å reconstruction is coloured according to the local resolution as estimated with ResMap46. c, The same set of particles that led to the 8.8 Å structure were also subjected to particle polishing and 3D focus classification on CSN (mask drawn as a blue line) with signal subtraction of CUL4A–DDB1. The data was divided into four classes revealing the dynamic of the CSN5–CSN6 heterodimer on cullin binding (Fig. 3a–c) (highlighted with a dashed red circle). Refinement of class I and class III led to a 7.8 Å and 8.8 Å resolution maps, respectively. d, Angular distribution of the particles included in the 6.4 Å (and 6.7 Å) data set. e, Gold-standard Fourier shell correlation curves (FSC) for the four refined models.

Extended Data Figure 4 The overall fold of each protein within the CSN–N8CRL4A complex.

ar, Unless specified, the individual subunits found in 6.4 Å CSN–N8CRL4A map are shown as cartoon representation. The cryo-EM density of each subunit is shown as a coloured mesh. CSN1 (a); CSN2 (b); CSN3 (c); CSN4 (d) CSN5 (8.8 Å resolution CSN–N8CRL4A map) (e) and CSN6 (8.8 Å resolution CSN–N8CRL4A map) (f); CSN7 (g); and CSN8 (h). Cryo-EM density and architecture of the CSN helical bundle found in the 6.7 Å CSN–N8CRL4A cryo-EM map (i); C-terminal (j) and N-terminal domain of CUL4A (k); RBX1 (l); DDB1 WD40 β-propeller domains BPA (m); BPB (n); and BPC (o); NEDD8 (N8) and WHB binding to CSN5 as shown in the 8.8 Å resolution CSN–N8CRL4A map (Fig. 1d) (p).

Extended Data Figure 5 Biophysical characterization of CSN mutants.

Determination of steady-state kinetics using the N8–PT22CRL4ADDB2 substrate. Initial rates observed following incubation of CSN or mutants (at 10 nM, unless specified otherwise) with increasing concentrations of N8–PT22CRL4ADDB2. The fit of the observed data to the Michaelis–Menten equation is shown in a, for 2 nM wild-type CSN (the experiment shown is a representative of four technical replicates (n = 2)); b, 20 nM CSN1279–527 (n = 3); c, 10 nM CSN4180–406 (n = 3). d, Dose response curve for the CSN–N8-Alexa488CRL4ADDB2 complex obtained by increasing concentration of unlabelled N8CRL4A (n = 3). e, As in d, but with increasing concentration of CRL4ADDB2 (n = 2). f, As in d, but with increasing concentration of unlabelled CSN (n = 2). g, Biotinylated CSN active site mutant (CSNASM) at 10 nM and terbium–streptavidin (Tb–streptavidin) conjugate (4 nM) were mixed with increasing concentrations of N8–Alexa488CRL4ADDB2 and the dissociation constants of CSN for N8–Alexa488CRL4ADDB2 obtained by fitting the TR-FRET signal assuming equimolar binding (see Supplementary Information). Three technical replicates are shown as overlaid curves to illustrate the reproducibility of the experiment. h, Binding of CSNASM (10 nM) and biotin-labelled ASM variants of CSN mutants at 10 nM (CSN (CSN2 (E33K, Y36A, K40D))), (CSN (CSN2 (R105E, K143D, R146E, K150D))), and (CSN (CSN2 (D266R, E267R))) to N8–Alexa488CRL4ADDB2 (n = 2). i, Steady-state kinetics for the CSN mutants (2 nM) used in h (n = 2). j, k, l, Binding of N8-Alexa488CRL4ADDB2 to CSNASM (10 nM) and biotin-labelled ASM variants of the CSN mutants at 10 nM (CSN (CSN2 (F259A, S296A, Q297A, E298K, K300D))) and (CSN (CSN4 (E165K, N169A, R170E))) (j) (n = 2); (CSN (CSN1 (R225E, H253A, Y257A, K260E))) (k) (n = 2); (CSN (CSN2 (D182K, D183K, E185K, D186R, D187K, K189D, K190E))) (l) (n = 2). m, n Steady-state kinetics for the CSN mutants (CSN (CSN71–218)) (2 nM) and (CSN (CSN730–218)) (2 nM) (m) (n = 2); 150 nM ((CSN(CSN6192-327)) (n = 2) and 2 nM (CSN (CSN1 (R225E, H253A, Y257A, K260E))) (n = 2) (n). In n, the deviation between fit and data for (CSN (CSN6192-327)) may suggest cooperative behaviour. o, Binding of the biotin-labelled ASM variant of the CSN mutant at 10 nM (CSN (CSN152–507, CSN31–400, CSN5 (S284A, F285A, L287A, H292A, R294D, K295D), CSN61–313)) to N8–Alexa488CRL4ADDB2 (n = 2). Data are technical replicates as indicated and shown as individual data points (for n ≤ 2) or mean ± s.d. (for n ≥ 3) in addition to the fit to the mean curve.

Extended Data Figure 6 Structural plasticity of CSN–CRL4 complexes.

a, The CUL4A C-terminal domain (CUL4ACTD) is coloured by conservation across all cullin families (automatically defined in ConSurf)47. Residues in proximity of the CSN2–CUL4ACTD interaction patches (shown as spheres) (Fig. 2a) are highly conserved. b, Superimposing WHA, α/β-domain and 4-HB domains (shown as transparent cartoon) of different structures (PDB accession codes 2HYE, 4A0K, 4P5O, 3DQV) reveal flexibility of the WHB domain. c, Superimposition of the CAND1–CUL4B–RBX1 structure (PDB accession code 4A0C, cyan) with the CUL4A–RBX1 conformation found in the cryo-EM CSN–N8CRL4A structures. d, The DDB1 inter-domain hinge shown in the close-up enables the DDB1 BPA and BPC subdomains to adopt different position with respect to DDB1 BPB allowing the CUL4A arm to swing at least 150°. The presence or absence of the substrate receptors does not affect the position of the cullin arm within CSN–N8CRL4A complexes. e, Rigid body dissection of apo-CSN. Boundaries (1) CSN1: 77–463; (2) CSN2: 30–180; (3) CSN2: 192–411; (4) CSN3: 3–345; (5) CSN4: 1–179; (6) CSN4: 180–298 (7) CSN4: 266–366; (8) CSN5: 24–249, CSN6: 29–296; (9) CSN7: 8–165; (10) CSN8: 11–167 (11) helical bundle: CSN1: 464–505, CSN2: 412–443, CSN3: 346–403, CSN4: 367–406, CSN5: 250–333, CSN6: 217–316, CSN7: 166–216, CSN8: 194–209. f, Structural comparison across 10 CSN crystallographic conformers, including P1 and P31 (PDB accession codes 4D10 and 4D18) crystals, provides evidence for large-scale conformational change. The models are coloured by levels of root mean squared deviation (RMSD). g, Superimposition of the PCI ring from apo-CSN (PDB accession code 4D18, grey) and the cullin-bound CSN conformation (Fig. 3d) (coloured as in Fig. 1). h, Superimposition of the helical bundle from apo-CSN (PDB accession code 4D18, grey) and in the cullin-bound CSN conformation showing structural rearrangement of the helical bundle on cullin binding (coloured as in Fig. 1).

Extended Data Figure 7 Receptor-induced remodelling of CSN.

a, Superimposition of atomic models for the CSN–N8CRL4ADDB2 (Fig. 1a) and CSN–N8CRL4A (Fig. 1b, grey). b, Schematic of the CSN subunits displacement shown in a, induced by the substrate receptor DDB2.

Extended Data Figure 8 Substrate binding inhibits CSN activity.

StrepII-tagged CSN was immobilized on Strep-Tactin beads and incubated with either CRL4ADDB2 (a) or N8CRL4ADDB2 (b). Following excessive washing, CRL4ADDB2 complexes remained bound to immobilized CSN (lane II). The immobilized complexes were subsequently incubated with 10 μM of a 31-bp THF oligo. Coomassie stained SDS–PAGE analysis revealed that CRL4ADDB2 and N8CRL4ADDB2 were dislodged in presence of DNA (lane IV) but not in a buffer control (lane IV buffer control in b). c, Increasing amounts of CRL4ADDB2 were mixed with a 16-bp Cy5-THF oligo and the dissociation constant Kd was determined by fitting the data to a model assuming one binding site (n = 2). d, Increasing concentrations of unlabelled 21-bp 6-4PP oligo were mixed with 16-bp Cy5-THF (100 nM) and CRL4ADDB2 (500 nM). The half-maximum effective concentration (EC50) was used to calculate the Ki (see Supplementary Information) inhibition constant (Ki is equivalent to Kd of 21-bp 6-4PP for CRL4ADDB2) (n = 2). e, Dose-response experiment with increasing concentration of lenalidomide titrated into CSN (2 nM) and N8–PT22CRL4ACRBN (500 nM) (n = 1). f, Binding of thalidomide–Cy5 to DDB1–CRBN (n = 1). g, Increasing concentration of lenalidomide–NEDD8 titrated into thalidomide–Cy5 (25 nM) and DDB1–CRBN (100 nM). EC50 was used to calculate Ki. These data indicate that lenalidomide–NEDD8 directly compete with thalidomide binding to DDB1-CRBN, and hence uses the same binding site (n = 1). h, i, Affinity of the biotin-labelled ASM variant of the CSN mutant (CSN (CSN1 (R225E, H253A, Y257A, K260E))) (10 nM) for N8CRL1SKP2 (n = 2) (h) and N8CRL3∆SPOP (i), determined by mixing increasing amounts of N8-Alexa488CRL1SKP2 and N8-Alexa488CRL3∆SPOP respectively with CSN (10 nM) and Tb-streptavidin (4 nM). Data were fitted as described in the Supplementary Information (n = 2). j, Steady-state kinetics of 2 nM CSN with a N8–PT22CRL4A substrate (n = 3). k, Binding curves for the biotin-labelled ASM variant of the CSN mutants (listed below the panel) to N8–Alexa488CRL4A (n = 2). One representative fit for each CSN mutant is shown from two technical replicates. l, Steady-state kinetics of CSN mutants (CSN (CSN2 (D182K, D183K, E185K, D186R, D187K, K189D, K190E))) and (CSN (CSN2 (F259A, S296A, Q297A, E298K, K300D))) with a N8–PT22CRL4ADDB2 substrate (n = 2). m, Steady-state kinetics of 2 nM CSN mutant (CSN (CSN4 (E165K, N169A, R170E))) with a N8–PT22CRL4ADDB2 substrate (n = 2). Data are technical replicates as indicated and shown as individual data points (for n ≤ 2) or mean ± s.d. (for n ≥ 3) in addition to the fit to the mean curve.

Extended Data Figure 9 Negative-stain single-particle EM reconstructions of CSN–N8CRL4ACRBN and CSN–N8CRL3∆SPOP.

a, Representative micrographs (scale bar, 50 nm; the whole data set includes 265 micrographs) and reference-free 2D class averages (scale bar, 10 nm) for CSN–N8CRL4ACRBN. b, Gold-standard Fourier shell correlation curve (FSC) for the CSN–N8CRL4ACRBN reconstruction. c, Angular distribution for CSN–N8CRL4ACRBN. d, CSN–N8CRL4ACRBN density fitted with DDB1–CRBN (PDB accession code 4CI1) and the cryo-EM CSN–N8CRL4A model (without DDB1). e, Representative micrographs (scale bar, 50 nm; the whole data set includes 104 micrographs) and reference-free 2D class averages (scale bar, 10 nm) for CSN–N8CRL3∆SPOP. f, Gold-standard FSC for the CSN-N8CRL3∆SPOP reconstruction. g, Angular distribution of the CSN–N8CRL3∆SPOP particles. h, Crystallographic models of CUL3NTD-BTB (PDB accession code 4EOZ), the substrate receptor MATH (PDB accession code 3HQI), and the NEDD8-engaged CSN cryo-EM model fitted in the CSN–N8CRL3∆SPOP negative-stain EM map. Modelling of the N-terminal MATH domain of SPOP (residues 28–166, Extended Data Fig. 1d) reveals its position within the CSN–N8CRL3∆SPOP architecture. i, Fit of the cullin-bound CSN model (Fig. 3d) and crystallographic models of N8CRL1SKP2/CKS1/p27 (PDB accession codes 1LDK, 3DQV, 2ASS) into the CSN–N8CRL1SKP2/CKS1 negative-stain map (EMDB accession code 2173). j, Fit of the cullin-bound CSN model (Fig. 3d) and crystallographic models of N8CRL1FBW7/CycE (PDB accession codes 1LDK and 2OVP) into the CSN–N8CRL1FBW7 negative-stain map (EMDB accession code 2174).

Extended Data Table 1 Determination of CSN affinities for N8CRLs, apo-CSN crystallographic data collection and refinement statistics

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Cavadini, S., Fischer, E., Bunker, R. et al. Cullin–RING ubiquitin E3 ligase regulation by the COP9 signalosome. Nature 531, 598–603 (2016) doi:10.1038/nature17416

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