Construct design, protein expression and purification

All proteins in this study are of human origin.

CUL9–RBX1 and variants: construct design and expression

Complementary DNA encoding full-length CUL9 (residues 1–2517) and RBX1 (residue 5 to C terminus) were cloned into pEG vectors individually, with CUL9 carrying a N-terminal TwinStrep-tag and consequent 3C protease cleavage site. Subsequently, gene expression cassettes were combined into a single pBIG1a vector73, which was used for bacmid generation from emBacY Escherichia coli. After introducing the bacmid into Sf9 insect cells (bought from Thermo Fisher, identifier no. 11496015) through transfection, the baculovirus was amplified and increased up to the third passage (P3). The resulting baculovirus-containing supernatant was then sterile filtered and used for infection of HEK293S cells. HEK293S GnTI− were bought from the American Type Culture Collection (ATCC) (identifier CRL-3022). HEK293S cells were grown to a density of around 3 × 106 cells per ml, infected with 10% (v/v) of virus and incubated for 16 h at 37 °C. Next, 10 mM sodium butyrate was added, the temperature was decreased to 30 °C and finally the cells were collected after 48 h (ref. 74).

CUL9 variants, either lacking selected residues or domains, or containing point mutations, were expressed by simultaneously infecting HEK293S cells with two separate baculoviruses. One baculovirus encoded the specific TwinStrep-tagged CUL9 variant, while the other carried the RBX1 gene.

To study the effects of selected CUL9 domains and sequences, the following residues were deleted in the listed CUL9 variants by replacing them with a GSGSGSGS linker:

CUL9∆CPH: 354–460

CUL9∆ARM9: 599–924

CUL9∆ARM3: 948–1105

CUL9∆DOC: 1167–1296

CUL9∆RING1: 2057–2142

CUL9ARM1dimer: 1652–1690

CUL9monomer: 1652–1690, and carries the following point mutations:

R125A and Y152A

CUL9 variants were also obtained by truncation of N- and/or C-terminal sequences. The following variants are truncated at the indicated residue(s):

CUL9∆ARIH-RBR: 1–1978

CUL9K1881R-∆ARIH-RBR: 1–1978 and carries the K1881R point mutation

CUL9ARIH-RBR: 1979–2517

Chimeric versions between CUL9 and CUL7 were generated by combining selected regions of both proteins:

CUL7CUL9-chimera: CUL7 residues 1–1208 (SBD to CR3) + CUL9 1538–1978 (4HB to WHB)

CUL9CUL7-chimera: CUL9 residues 1–1537 (SBD to CR3) + CUL7 1209–1698 (4HB to WHB)

All CUL9 variants were coexpressed with RBX1 except for CUL9ARIH-RBR as this only encompasses the ARIH-RBR sequence, which does not bind RBX1.

Protein purification of CUL9–RBX1 and variants

HEK293S cells were collected by centrifugation and resuspended in lysis buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 1 mM dithiothreitol (DTT), 1× cOmplete Protease Inhibitor, Roche). Subsequently, cells were lysed via sonication and centrifuged for 30 min at 20,000g. The protein-containing supernatant was incubated with Strep-Tactin resin for 30 min at 4 °C. After transferring the resin to gravity flow columns, five washing steps were performed using wash buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM DTT). The protein was eluted with 25 mM HEPES pH 7.5, 200 mM NaCl, 1 mM DTT, 2.5 mM Desthiobiotin. Next, Strep-tagged fusion protein was cleaved by incubation with 3C protease (molar ratio 1:50 protease:protein) for 2–3 h at room temperature and purified by size-exclusion chromatography on a Superose 6 Increase 10/300 GL column (GE Healthcare) using 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. The elution volume of the protein complex was close to the Superose 6 void volume and indicated formation of a larger assembly.

Expression and purification of TP53

His-lipoyl domain-tagged TP53 was expressed in E. coli BL21 Rosetta and induced with isopropyl beta-d-thiogalactoside (IPTG) (0.5 mM) at an optical density of 0.6–0.8 and expression continued at 18 °C overnight. E. coli cells were disrupted via sonication and cell lysate subjected to centrifugation at 20,000g for 30 min. Target protein-containing supernatant was subjected to immobilized metal affinity chromatography. After elution with imidazole, fusion protein was dialyzed overnight at 4 °C with tobacco etch virus (TEV) protease (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT, 1:50 molar ratio target protease:protein). Size-exclusion chromatography in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT on a Superose 6 Increase 10/300 GL column (GE Healthcare) was used to purify tetrameric TP53 away from tag and protease. The purified TP53 was used as a substrate in ubiquitylation assays.

Expression and purification of CSN

CSN constructs for insect cell expression were generated using the biGBac system73. Here, CSN3 features a C-terminal 3C cleavage site, succeeded by a 3× Strep-tag, while CSN5 carries an N-terminal 6× His-tag with subsequent 3C site. All other subunits including CSN1, CSN2, CSN4, CSN6, CSN7b and CSN8 were untagged. These CSN subunits 1–8 were coexpressed from a single baculovirus in Trichoplusia ni High-Five insect cells. Insect cells were collected and lysed as described for HEK293S cells and the protein complex-containing supernatant was incubated with Strep-Tactin beads. After washing and elution, immobilized metal affinity chromatography (Ni-NTA) was performed and affinity tags were cleaved overnight while dialyzing in 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 3C protease in 1:50 molar ratio. Finally, size-exclusion chromatography was carried out to buffer exchange into in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT.

Expression and purification of GST-tagged proteins: ubiquitin, NEDD8, UBA1, E2 enzymes, CUL1–RBX1, CUL5–RBX2, ARIH1, ARIH2 and SENP8

Full-length human CUL1 and CUL5 were coexpressed with their respective RING protein GST-TEV-RBX1 (residue 5 to C terminus) or GST-TEV-RBX2 (residue 5 to C terminus) in Trichoplusia ni High-Five insect cells. GST-TEV-UBA1 was also expressed in insect cells. Full-length UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2M, UBE2N, UBE2Q2, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE2V1, UBE2V2, SENP8, NAE1-UBA3, ARIH1, ARIH2 and mutant versions (the so-called OPEN mutants that mutationally release autoinhibitory Ariadne-Rcat domain interactions, F430A E431A E503A for ARIH1, and L381A E382A E455A for ARIH2) cloned into pGEX-4T1 vectors were expressed as GST-TEV fusion proteins in E. coli Rosetta 2 (DE3). Expression was induced with IPTG (0.1 mM for ARIH1 and ARIH2, 0.5 mM for E2s, Ubiquitin, NEDD8 and SENP8) at an optical density of 0.6–0.8. For ARIH1 and ARIH2, 0.1 mM ZnCl2 was added to the TB medium. Expression continued overnight at 18 °C for all proteins. Cell lysates containing GST-fusion proteins were subjected to disruption via sonication. Following centrifugation, protein-containing supernatant was then allowed to incubate with glutathione sepharose beads. The beads were washed several times with wash buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM DTT). Cleavage with TEV protease was performed on beads overnight. After elution of cleaved protein from the column with wash buffer, the target protein was subjected to ion exchange and size-exclusion chromatography in a final buffer of 25 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP)7,9,10,46,75.

Expression and purification of APEX2

C-terminal 3× FLAG-tagged APEX2 was expressed analogously to CUL9 in HEK293S cells. Cell lysate was incubated for 1 h with anti-FLAG M2-affinity gel, washed five times with buffer (25 mM HEPES pH 7.5, 150 mM NaCl) and eluted with the same buffer including 100 ng ml−1 FLAG-peptide. Eluted protein was subjected to ion exchange, concentrated and buffer exchanged into 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT before usage as a substrate in ubiquitylation assays.

Expression and purification of ubiquitin

WT ubiquitin was produced in E. coli Rosetta 2 (DE3) cells and subsequently purified without the use of a tag. The purification process involved a glacial acetic acid purification step76, followed by ion exchange using an S-column and subsequent size-exclusion chromatography. In short, acetic acid was slowly added to the bacterial lysate until a pH of ~4.5 was reached. This precipitated most proteins other than ubiquitin. After dialysis into 25 mM sodium acetate pH 4.5, 100 mM NaCl, the dialyzed ubiquitin was centrifuged and cleared supernatant was subjected to ion exchange chromatography on a S-column, followed by size-exclusion chromatography on an SD75 10/300 GL column (GE Healthcare) into 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT to yield WT, tagless ubiquitin.

Cryo-EMSample preparation and data collection for CUL9–RBX1

After size-exclusion chromatography, peak fractions of CUL9–RBX1 were pooled and concentrated to 5 mg ml−1. The protein complex was then cross-linked in batch with 0.05% (v/v) Glutaraldehyde for 10 min at room temperature, followed by the addition of and incubation with 50 mM Tris pH 7.5 for another 5 min to quench the cross-linking reaction. Shortly before plunging, fluorinated Fos-Choline-8 (Anagrade) was added to the protein sample at a final concentration of 1.5 mM. This was essential to overcome preferred orientation of the sample. Subsequently, holey carbon grids (Quantifoil, R1.2/1.3, 200 mesh) were glow discharged, and 3 μl of CUL9–RBX1 was applied to the grid at 95% humidity and 4 °C using a Vitrobot Mark IV (Thermo) and plunge-frozen into liquid ethane (blot force 3, blot time 3 s). After several screening datasets to identify the ideal compromise between ice thickness and particle density, high-resolution data were collected on a Titan Krios transmission electron microscope (TEM), equipped with a post-GIF Gatan K3 Summit direct electron detector in counting mode. Datasets were collected using SerialEM (v.3.8.0-b5) and FEI EPU (v.2.7.0). Videos were collected at a nominal magnification of ×105,000, equaling 0.8512 Å/pixel at the specimen level. The target defocus ranged between −0.7 and −2.8 μm and the total dose of ~60 e/Å2 was distributed over 40 frames.

Processing of CUL9–RBX1 cryo-EM data

Motion-correction and dose weighting were performed using RELION v.3.1 (ref. 77) and the contrast transfer function (CTF) was estimated using CTFFIND-4.1 (ref. 78). Particles were picked using Gautomatch (v.0.56) (K. Zhang, MRC Laboratory of Molecular Biology) with reference-free blob-based picking. Then, 16,800 micrographs with a maximum resolution estimate better than 5 Å were imported into RELION v.3.1 (ref. 77), from which ~1.21 million particles were extracted applying 5.25× binning. These were subjected to several rounds of 3D classification, followed by initial model generation and 3D classification. After 3D classification with and without masking, several sets of particles for the hexamer, the cullin dimer or the E2-density map were re-extracted at full pixel size. Subsequently, masked 3D auto-refinement, CTF-refinement and particle polishing resulted in maps with resolutions of less than 5 Å. RELION77 postprocessing and DeepEMhancer (v.2020.09.07)79 were used for sharpening of the final maps. A higher-resolution map of the inactive (unneddylated) cullin dimer complex was obtained by performing symmetry expansion following the application of C3 symmetry during the previous refinement. During 3D classification, particles with the neddylated conformation were excluded. Local refinement resulted in a 3.37 Å map of the cullin dimer, where both protomers are in the inactive (unneddylated) conformation. Unfortunately, symmetry expansion while selecting for the neddylated (active) conformation did not yield substantially more particles or result in map improvement.

Model building and refinement

Coordinates were built for three complexes, in the following sequence: the mixed cullin dimer between neddylated and unneddylated CUL9–RBX1 using the map shown in Fig. 2c, a hexamer using the map shown in Fig. 1b, and an unneddylated CUL9–RBX1 dimer using the map shown in Extended Data Fig. 3c.

The cullin dimer structure comprising neddylated and unneddylated CUL9–RBX1 also contains an E2 and was built as follows. A structural model of CUL9, predicted by Alphafold2 (ref. 39), was split into several domains and segments that were fit into the cryo-EM map using Chimera (v.1.13.1)80. Models for most domains could be initially docked in the well-defined secondary structure, with the exception of the CUL9 CPH, ARM9, DOC and Rcat domains, which could not be placed in the map. The CUL9 Ariadne domain was clearly resolved in both the neddylated and unneddylated protomers, albeit in different relative orientations. The remaining regions of the CUL9 ARIH-RBR element were only resolved in the neddylated protomer. RBX1 was built based on the CRL7FBXW8 structure21. The NEDD8 linked to CUL9 was built based on the structure representing ubiquitin transfer from UBE2L3 to ARIH1 bound to a neddylated CUL1-based CRL9. That structure also provided coordinates for UBE2L3 docked into the density for an E2 bound to the CUL9 ARIH-RBR element RING1 domain. UBE2L3 was used for E2 in the structure based on ubiquitylation assays, AP–MS, XL–MS and isothermal calibration (ITC) binding experiments. Ultimately, the model was completed by iterative cycles of manual rebuilding and refinement using Coot (v.0.8.9.1)81, alternating with real-space refinements with Phenix.refine (v.1.17.1)82. For lower resolution parts of the map, side-chains were removed, including wholesale removal of side-chains across the CUL9 ARM1-3 domains, NEDD8, and the E2 (UBE2L3). For the RBX1 RING domain, the side-chain placement was maintained from the starting model from the complex with CUL7 (ref. 21). The coordinates for this cullin dimer complex (comprising unneddylated CUL9–RBX1 and E2-bound neddylated CUL9–RBX1) served as the starting model for the other two structures.

The cryo-EM map of the full hexamer did not allow placement of side-chains but most domains other than the ARIH-RBR element and NEDD8 were clearly visible on a secondary structure level. The structure of the dimeric complex between unneddylated and neddylated CUL9–RBX1—without the ARIH-RBR element and NEDD8—was fit into the hexameric cryo-EM map three times using Chimera (v.1.13.1)80. Clear density was observed for the three unneddylated protomers, while the CUL9 WHB domain RBX1 RING domain from the alternating protomers were poorly resolved and thus these regions were removed from the coordinate file. Side-chains and remaining unresolved segments were removed in Coot81, and the structure was finalized by rigid body refinement with Phenix.refine (v.1.17.1)82. It seems likely that relatively lower resolution of the CUL9 WHB domain and RBX1 RING domain in some protomers results from intrinsic conformational heterogeneity and/or a mixture of neddylated and unneddylated complexes. To represent both versions in a single hexamer, the position of the RING domain from the neddylated CUL9–RBX1 complex was shown for alternating protomers in Fig. 1c.

To obtain the structure of the unneddylated cullin dimer, the coordinates for the unneddylated CUL9 protomer, bound to the N-terminal strand from RBX1, from the dimer described above were fit using Chimera (v.1.13.1)80 into the cryo-EM map obtained by symmetry expansion. Side-chains were remodeled using Coot (v.0.8.9.1)81, followed by real-space refinement with Phenix.refine (v.1.17.1)82. The RBX1 RING domain from the published complex with CUL7 (ref. 21) was wholesale docked into remaining density, and the final model was polished by rigid body refinement with Phenix.refine (v.1.17.1)82.

Sample preparation for CUL9∆CPH–RBX1, CUL9∆ARM9–RBX1, CUL9∆ARIH-RBR–RBX1 and CUL9∆DOC–RBX1 and data collection

CUL9∆CPH–RBX1, CUL9∆ARM9–RBX1, CUL9∆ARIH-RBR–RBX1 and CUL9∆DOC–RBX1 were purified following the same protocol as for WT CUL9–RBX1, concentrated to 3 mg ml−1 and snap frozen in liquid nitrogen for storage. Before plunging, samples were thawed on ice and centrifuged for 10 min at 4 °C, 14,000g. Cross-linking and plunging was performed as for WT CUL9–RBX1. Datasets were collected either on an Arctica TEM equipped with a Falcon III electron detector in linear mode, or on a Glacios TEM equipped with K2 Summit direct detector in counting mode. Videos were captured using the Arctica TEM with a nominal magnification of ×73,000, resulting in a pixel size of 1.997 Å/pixel at the specimen level. Alternatively, videos were recorded on the Glacios TEM with a nominal magnification of ×22,000, yielding a pixel size of 1.885 Å/pixel at the specimen level. The intended defocus spanned from −1.2 to −3.3 µm, and the cumulative exposure, approximately 60 electrons per Å2, was distributed across 40 frames.

Processing of cryo-EM data for CUL9∆CPH–RBX1, CUL9∆ARM9–RBX1, CUL9∆ARIH-RBR–RBX1 and CUL9∆DOC–RBX1

Motion-correction and dose weighting were performed using RELION 4.0 (ref. 77) and the CTF was estimated using CTFFIND-4.1 (ref. 78). Particles were picked using Gautomatch (v.0.56) (K. Zhang, MRC Laboratory of Molecular Biology) with reference-free blob-based picking. All datasets used the hexameric CUL9–RBX1 template as reference for initial 3D classification without application of symmetry, followed by several iterations of 3D refinement and alignment-free 3D classification. Finally, clean particle sets were unbinned and refined, followed by PostProcessing in RELION77. Cryo-EM maps were analyzed in ChimeraX (v.1.2.5).

Assays to assess ubiquitylation, neddylation and deneddylationFluorescent labeling of ubiquitin and NEDD8

Ubiquitin and NEDD8 were expressed with an additional N-terminal cysteine. This cysteine was ultimately used to label the proteins fluorescently. After size-exclusion chromatography into 25 mM HEPES, pH 7.5, 150 mM NaCl and 5 mM DTT, protein was desalted twice with Zeba Spin Desalting columns to remove DTT as it would be interfering in the reaction with the maleimide. Next, fluoresceine-5-Maleimide (dissolved in anhydrous dimethylsulfoxide (DMSO)) was incubated with ubiquitin with a tenfold molar excess. The overall concentration of DMSO did not surpass 5% in the reaction. This mixture was then incubated at room temperature for 2 h before the addition of 10 mM DTT to halt the reaction. The reactions were desalted to eliminate any remaining unreacted maleimide. Subsequently, the reaction mixture underwent two rounds of size-exclusion chromatography into 25 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM DTT, yielding fluorescent ubiquitin (*Ub) or NEDD8 (termed *N8 or *NEDD8 in figures and text).

Ubiquitylation assays

All ubiquitylation reactions were performed in a multi-turnover format. Assays screen CUL9–RBX1-dependent ubiquitylation activity with a range of E2 enzymes, and compare activities of WT CUL9–RBX1 and E2 UBE2L3 versus variants toward TP53 and APEX2 substrates. Ubiquitylation assays were prepared by mixing 0.25 μM UBA1, 1 μM E2, 1 μM E3 (WT or variant), 15 µM *Ub, with or without 1 µM substrate (TP53 or APEX2) in 25 mM HEPES pH 7.5, 100 mM NaCl, 2.5 mM MgCl2. The reaction was started by addition of 2.5 mM ATP, incubated at room temperature and quenched at indicated time points with SDS–PAGE sample buffer. SDS–PAGE gels were imaged with an Amersham Typhoon Imager (Cy2 channel) to visualize fluorescently labeled ubiquitin.

Neddylation assays

Posttranslational modification of cullins with NEDD8 on their respective WHB domains was assayed in the same format as for ubiquitylation reactions. For this purpose, 0.5 μM NAE (NAE1-UBA3), 1 μM E2 (either UBE2F or UBE2M), 1 µM cullin (CUL1–RBX1, CUL5–RBX2 or CUL9–RBX1 or variants thereof) and 5 µM *NEDD8 were mixed in 25 mM HEPES pH 7.5, 150 mM NaCl, 2.5 mM MgCl2 and reaction was started by addition of 2.5 mM ATP (final concentration). The reactions were quenched at the indicated time points with SDS–PAGE sample buffer, subjected to SDS–PAGE and analyzed using an Amersham Typhoon Imager (Cy2 channel) to visualize fluorescent NEDD8. Alternatively, if neddylation reaction were to be subsequently used to study deneddylation of neddylated cullin by either CSN or SENP8, reactions were quenched with either 10 mM DTT (in the case of CSN) or 15 mM EDTA (for SENP8).

Deneddylation assays

NEDD8 modification on canonical cullins is specifically removed by the deneddylase CSN. In contrast, SENP8 is a deneddylase responsible for proteolytic cleavage of pro-NEDD8, hyper-neddylated cullins and other proteins. To investigate whether NEDD8 modification on CUL9 could be removed by either of the deneddylases, 2 μM SENP8 or 0.1 μM CSN was added to the quenched neddylation reactions. The deneddylation reactions were stopped at the indicated time points by addition of SDS–PAGE sample buffer and SDS–PAGE gels were imaged with an Amersham Typhoon Imager (Cy2 channel) to visualize Fluorescein-labeled NEDD8.

Biochemical and biophysical characterization of CUL9–RBX1 and variantsMass photometry

WT CUL9–RBX1 was analyzed using mass photometry to estimate size and oligomeric state. Calibration was performed by using a protein mixture containing a variety of molecular masses, including Aprotinin, Ribonuclease A, Carbonic anhydrase, Ovalbumin, Conalbumin and Blue dextran, all present at a final concentration of approximately 20 nM for each component. WT CUL9–RBX1 was measured in a final concentration of 50 nM in 25 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP. Data were collected over 50 frames and 30 s on a Refeyn TwoMP mass photometer using Refeyn AcquireMP v.2.3.0. Data were analyzed with the Refeyn DiscoverMP v.2.3.0 software.

SEC–MALS

In addition to mass photometry, SEC–MALS analysis was performed to estimate the molecular weight of the CUL9–RBX1 complex. For this purpose, 70 µl of purified protein at 3 mg ml−1 was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) connected to a DAWN8 + TREOS MALLS detector and Optilab rEX differential refractometer (Wyatt Technologies). Each run was performed at a flow rate of 1 ml min−1 in 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP at room temperature. Molecular-weight calculations were performed with ASTRA software v.5.3 (Wyatt Technologies).

Size-exclusion chromatography of CUL9–RBX1 variants

Size-exclusion chromatography was used to examine the oligomeric status of CUL9–RBX1 and CUL9–RBX1 variants containing mutations designed to disrupt the dimerization interfaces (Fig. 4b and Extended Data Fig. 1a). To establish a reference, a size standard mixture (Bio-Rad), containing thyroglobulin, γ-globulin, ovalbumin, myoglobin and vitamin B12 was loaded onto a Superose 6, 5/150GL column (GE). Subsequently, a 50 μl sample of 1.5 μM WT or variant CUL9–RBX1 was loaded onto the Superose 6, 5/150GL column (GE). The gel filtration buffer contained 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT for all runs.

Sucrose gradients

To study endogenous CUL9 assemblies, sucrose gradient fractionation was performed. For this, 1 mg of total protein cell lysate was loaded onto a continuous 5–40% sucrose gradient (weight and volume in 25 mM HEPES 7.5, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.05% TWEEN and 1× cOmplete Protease Inhibitor Mix), which was generated via gradient maker (Biocomp Gradient Master 108). Samples were centrifuged in an ultracentrifuge (Thermo Scientific Sorvall WX+ Ultracentrifuge) equipped with a SW60Ti rotor at 160,000g for 16 h at 4 °C. Fourteen 300 µl fractions were collected from the top of the gradient, separated by SDS–PAGE and followed by immunoblotting using indicated antibodies. The blots were developed using Clarity Western ECL Substrate (Bio-Rad, catalog no. 16640474) and imaged using an Amersham Imager 600 (GE Lifesciences). Bio-Rad’s Gel Filtration Standard and purified WT hexameric CUL9–RBX1, as well as dimeric CUL9R125A Y152A–RBX1 were run for comparison. Endogenous CUL9 samples were run in triplicate and distribution of CUL9 protein over the fraction was plotted, normalized to the total CUL9 protein amount in all fractions.

ITC analysis

ITC measurements were performed on a MicroCal PEAQ-ITC (Malvern) at 25 °C with a setting of 19 × 2 μl injections. CUL9ARIH, UBE2D2, UBE2D3 or UBE2L3 were all dialyzed into dialysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, and 0.5 mM TCEP) before analysis. For measurements, the syringe contained a concentration of E2 enzyme at 300–500 μM and the cell contained CUL9ARIH-RBR at 25 μM. The heats of dilution for diluting E2s into measurement buffer were subtracted from binding experiments before curve fitting. Manufactured supplied software was used to fit the data to a single-site binding model and to determine the stoichiometry (N), the molar reaction enthalpy ΔH, the entropy change ΔS and the association constant Ka. The dissociation constant, KD, was calculated from 1/Ka.

Cell culture and cell treatmentsU2OS cell culture

U2OS cells (ATCC HTB-96)17 were maintained in McCoy’s 5A medium (Gibco) supplemented with 10% fetal calf serum (Gibco), 100 U ml−1 penicillin, 0.1 mg ml−1 streptomycin (Gibco) at 37 °C, 5% CO2. For better detection, CUL9 was subcloned into pcDNA5 FRT/TO vector with HA tag at the C terminus. To ensure this was the only CUL9 present, U2OS ∆CUL9 cells17 were transiently transfected using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s protocol and incubated for 48–96 h at 37 °C before performing further analysis.

Immunoblot analysis of cell lysates

The cells were gathered by centrifugation at 360g, washed once with ice-cold 1× PBS, and resuspended in lysis buffer (25 mM HEPES 7.5, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.05% TWEEN and 1× cOmplete Protease Inhibitor Mix (Roche)), supplemented with 0.01% TWEEN and incubated on ice for 10 min. Cells were homogenized by douncing ten times. The obtained lysates were cleared by centrifugation at 23,000g for 30 min at 4 °C, and protein concentration was determined by Micro BCA-Protein Assay (Thermo Scientific, catalog no. 23235). For immunoblot analysis, lysates were denatured with SDS sample buffer, boiled at 95 °C for 5 min, separated on SDS–PAGE and proteins were visualized by immunoblotting using indicated primary antibodies: NEDD8 (CST, 2745), CUL9 specific antibody was a gift from A. Alpi, Vinculin (Abcam, catalog no. ab129002) and β-Actin (CST, catalog no. 4967). All primary antibodies in this paper were used at a final concentration of 1 μg ml−1.

U2OS cell treatment with MLN4924 and CSN5i-3

MLN4924 inhibits NAE enzyme and thus neddylation in cells55. CSN5i-3 is an inhibitor that targets the catalytic CSN5 subunit of the CSN, and prevents deneddylation of canonical cullins56. To test how both inhibitors affect CUL9, U2OS parental and ∆CUL9 knock-out cells were treated with either 0.5 µM MLN4924 (also known as Pevonedistat, Selleckchem, S7109) or 3 µM CSN5i-3 (MCE, HY-112134). Cell viability and confluency were carefully monitored throughout the incubation period, ensuring the confluency remained between 40 and 90%. Control cells were treated with DMSO. After 24 h of drug treatment, cell lines were transiently transfected with HA-tagged CUL9 using Lipofectamine 3000 (Thermo Fisher) following the manufacturer’s protocol and incubated for further 24 h before cell lysis and immunoblot analysis.

siRNA knockdown of UBE2F and UBE2M

U2OS cells were seeded at a density of 30–40% cells per well, ensuring approximately 70–80% confluence on the day of transfection. Small-interfering RNAs (siRNAs) were obtained from Dharmacon. For siRNA knockdown, the cells were transfected with 40 pmol siRNA targeting UBE2F (CAAGUAAACUGAAGCGUGA, AUGACUACAUCAAACGUUA, CAAUAAGAUACCCGCUACA, CUGAAGUUCCCGAUGCGUA, catalog numbers J-009081-09, J-009081-10, J-009081-11 and J-009081-12), UBE2M (GAAAUAGGGUUGGCGCAUA, AAGCCAGUCCUUACGAUAA, UUAAGGUGGGCCAGGGUUA, GAUGAGGGCUUCUACAAGA, J-004348-05, J-004348-06, J-004348-07 and J-004348-08) or nontargeting (UGGUUUACAUGUCGACUAA) using RNAi Max (Thermo Fisher, 13778075) according to manufacturer’s protocol. The transfected cells were incubated at 37 °C for 48 h, after which the cells were lysed and knockdown efficiency was assessed by immunoblotting.

Co-immunoprecipitation in U2OS cells

HA-tagged proteins were captured from 1 mg total cell lysate using anti-HA affinity matrix (Pierce, catalog no. 88836) overnight at 4 °C. All immunoprecipitation reactions were washed in lysis buffer, and immunoadsorbed proteins were eluted by boiling in reducing SDS sample buffer, separated by SDS–PAGE followed by immunoblotting using indicated antibodies.

Mass spectrometry analysesXL–MS Sample preparation

Purified WT CUL9–RBX1 or the monomeric variant were cross-linked at a concentration of 4 µM protein complex with 2 mM bis(sulfosuccinimidyl)suberate for 20 min at room temperature. Cross-linking was quenched by adding 50 mM Tris-HCl pH 7.5 (final concentration) and incubated for 5 min. Cross-linked proteins were denatured, reduced and alkylated by addition of 4 M urea, 40 mM 2-cloroacetamide (Sigma-Aldrich) and 10 mM TCEP (Thermo Fisher Scientific) in 50 mM Tris-HCl. After incubation for 20 min at 37 °C, the samples were diluted 1:3 with mass spectrometry grade water (VWR) and proteins were digested overnight at 37 °C by addition of 0.5 µg of LysC and 1 µg of trypsin (Promega). Thereafter, the solution was acidified with trifluoroacetic acid (TFA) (Merck) to a final concentration of 1%, followed by desalting of the peptides using Sep-Pak C18 1cc vacuum cartridges (Waters).

Data acquisition LC–MS analysis

Peptides were dissolved in buffer A (0.1% formic acid) and 1/20 of the peptides were analyzed by liquid chromatography with tandem mass spectrometry (LC–MS/MS) comprising an Easy-nLC 1200 (Thermo Fisher Scientific) coupled to an Exploris 480 or a QExactive HF mass spectrometer (Thermo Fisher Scientific). Peptides were separated within 60 min on a 30 cm analytical column (inner diameter 75 μm; packed in-house with ReproSil-Pur C18-AQ 1.9 μm beads, Dr. Maisch GmbH) using a gradient of buffer A to buffer B (80% acetonitrile (ACN), 0.1% FA). The mass spectrometer was operated in data-dependent mode and specialized settings for the data acquisition of cross-linked peptides were set: we have used higher-energy C-trap dissociation with normalized collision energy values of 19, 27 and 35, and we have excluded charge state 2 from being fragmented to enrich the fragmentation scans for cross-linked peptide precursors.

Data processing XL–MS

The acquired raw data were processed using Proteome Discoverer (v.2.5.0.400) with the XlinkX/PD nodes integrated83. The database search was performed against a FASTA file containing the sequences of the proteins under investigation. Disuccinimidyl suberate was set as a cross-linker. Cysteine carbamidomethylation was set as fixed modification and methionine oxidation and protein N-terminal acetylation were set as dynamic modifications. Trypsin/P was specified as protease and up to two missed cleavages were allowed. Identifications were only accepted with a minimal score of 40 and a minimal delta score of 4. Filtering at 1% false-discovery rate at the cross-link spectrum match (CSM) and cross-link level was applied. The data were analyzed with cross-link analyzer v.1.1.4.

AP–MS Expression and pulldown

CUL9–RBX1 and variants were expressed as described above but in triplicates and the Strep-pulldown elutions were subsequently processed for mass spectrometry.

Sample preparation

For the reduction and alkylation of the proteins, 100 µl of SDC buffer (1% sodiumdeoxycholate, 40 mM 2-chloroacetamide (Sigma-Aldrich), 10 mM TCEP (PierceTM, Thermo Fisher Scientific) in 100 mM Tris-HCl, pH 8.0) was added and the proteins were incubated for 20 min at 37 °C. The samples were diluted 1:2 with water and digestion proceeded overnight at 37 °C by addition of 0.5 µg of trypsin (Promega). The solution of peptides was then acidified with TFA (Merck) to a final concentration of 1% followed by purification via SCX StageTips. Samples were vacuum dried and resuspended in 12 µl of buffer A (0.1% formic acid (Roth) in mass spectrometry grade water (VWR)).

LC–MS/MS data acquisition

Here, 800 ng of the desalted peptide mixture was separated on an analytical column (30 cm, 75 µm inner diameter, packed in-house with ReproSil-Pur C18-AQ 1.9 µm beads, Dr. Maisch GmbH) by an Easy-nLC 1200 (Thermo Fisher Scientific) at a flow rate of 250 nl min−1 while heating the column to 60 °C. The LC was coupled to a QExactive HF mass spectrometer (Thermo Fisher Scientific). As a LC-gradient, the following steps were programmed with increasing addition of buffer B (80% ACN, 0.1% formic acid): linear increase from 7 to 30%B over 60 min, followed by a linear increase to 60%B over 15 min, then followed by a linear increase to 95%B and finally, the percentage of buffer B was maintained at 95% for another 5 min.

The mass spectrometer was operated in a data-dependent mode with survey scans from 300 to 1,650 m/z (resolution of 60,000 at m/z = 200), and up to ten of the top precursors were selected and fragmented using higher-energy collisional dissociation (with a normalized collision energy of value of 28). The MS2 spectra were recorded at a resolution of 15,000 (at m/z = 200). AGC target for MS1 and MS2 scans were set to 3 × 106 and 1 × 105, respectively, within a maximum injection time of 100 and 60 ms for MS1 and MS2 scans, respectively.

Data analysis

Raw data were processed using the MaxQuant computational platform (v.2.2.0.0)84 with standard settings applied. The peak list was searched against the Human UniProt database (SwissProt and TrEMBL) with an allowed precursor mass deviation of 4.5 ppm and an allowed fragment mass deviation of 20 ppm. Cysteine carbamidomethylation was set as static modification, and methionine oxidation and N-terminal acetylation as variable modifications. The match between-run option was enabled, and proteins were quantified across samples using the label-free quantification algorithm in MaxQuant generating label-free quantification intensities.

Mass spectrometric analysis of ubiquitylation sites on CUL9–RBX1 and TP53 Sample preparation

Here, 4 µM CUL9–RBX1 was incubated with 0.2 µM UBA1, 4 µM UBE2L3, 40 µM WT ubiquitin and 2.5 mM MgATP with or without TP53 for 30 min at room temperature. The reactions were quenched with 10 mM DTT and 6 µg of total protein amount was alkylated, reduced and digested simultaneously using 1 M urea in 50 mM ABC with 10 mM TCEP, 40 mM 2-chloracetamide and 0.5 µg of trypsin (Sigma-Aldrich) at 37 °C overnight with agitation (1,500 rpm) on an Eppendorf Thermomixer C. SDB-RPS (Empore) StageTips were used for peptide desalting. In brief, peptides were diluted using a 1:10 ratio (peptide, 1% TFA in isopropanol), loaded to StageTips and washed with 200 µl of 1% TFA in isopropanol and then with 0.2% TFA/2% ACN twice. Peptide elution was done using 75 µl of 80% ACN/1.25% NH4OH. Samples were then dried using a SpeedVac centrifuge (Concentrator Plus; Eppendorf) for 1 h at 30 °C and subsequently resuspended 0.2% TFA/2%. Finally, 50 ng of peptides were injected into LC–MS/MS.

Data-dependent acquisition LC–MS analysis

For LC–MS/MS analysis, we used the following setup: 50 cm reversed phase column (75 μm inner diameter, packed in-house with ReproSil-Pur C18-AQ 1.9 μm resin), a homemade oven that maintained a column temperature constant at 50 °C, an EASY-nLC 1200 system (Thermo Fisher Scientific) connected online to the mass spectrometer (Orbitrap Exploris 480, Thermo Fisher Scientific) via a nano-electrospray source. For peptide separation we used a binary buffer system (buffer A, 0.1% formic acid and buffer B, 80% ACN, 0.1% formic acid). Peptides were eluted using a 60 min gradient with a constant flow rate of 300 nl min−1. The gradient starts at 3% buffer B and increases to 8% after 35 min, 36% after 40 min, 45% after 44 min and 95% after 48 min until it stays constant until 52 min and decreases to 5% buffer B after 60 min. The following settings were used for mass spectrometry data acquisition: data-dependent acquisition mode with a full scan range of 250–1,350 m/z, 60,000 resolution, 3 × 106 automatic gain control (AGC), 20 ms maximum injection time and 28 higher-energy collision dissociation. Every survey scan was followed by 12 data-dependent acquisition scans with a 30,000 resolution, a 1 × 106 AGC and a 110 ms maximum injection time.

Data processing and bioinformatics analysis

Raw files were process using MaxQuant v.1.6.2.10 (ref. 84). For the search we used a human UniProt FASTA file with 42,347 entries. The digestion mode was set to trypsin/P with a maximum of two missed cleavage sites and maximum and minimum peptide lengths of 25 and 8, respectively. Variable modifications were set to oxidation (M), acetyl (Protein N-term) and GlyGly (K) and fixed modification were set to carbamidomethylation (C). Match between run was enabled. The bioinformatics analyses were done using Python v.3.5.5 with the following packages: numpy v.1.21.5, and pandas v.1.4.2.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.