Protein purification and complex assembly
Seven of the eight human CSN subunits, excluding CSN5, were overexpressed and purified from Escherichia coli (E. coli). Two trimeric subcomplexes, CSN1–2–3 and CSN4–6–7, were individually purified through co-expression. For the CSN1–2–3 subcomplex, CSN2 was subcloned into a modified pGEX4T1 vector (Amersham Biosciences) containing an N-terminal glutathione S-transferase (GST) tag followed by a tobacco etch virus (TEV) protease cleavage site. Truncated CSN3 (residues 1–409) and full-length CSN1 were individually subcloned into a modified pET15b vector (Novagen) containing an N-terminal His tag with a TEV protease cleavage site. The expression cassettes of CSN1 and CSN31–409—each driven by a T7 promoter and terminated by a T7 terminator—were subsequently inserted into the pGEX4T1-CSN2 plasmid. The resulting construct contained three independent expression cassettes for CSN2, CSN1 and CSN31–409, respectively. The CSN1–2–3 complex was co-expressed in E. coli BL21(DE3) cells (Novagen), purified by glutathione-affinity chromatography and subjected to TEV protease cleavage to remove the GST tag. The cleaved complex was further purified by anion-exchange (Source Q, Cytiva) and size-exclusion chromatography (Superdex 200, Cytiva). The CSN4–6–7 subcomplex was prepared using the same strategy. In this case, truncated CSN7b (residues 1–239) was subcloned into the modified pGEX4T1 vector, whereas full-length CSN4 and CSN6 were inserted into the modified pET15b vector. CSN8 was subcloned into a modified pET15b vector encoding an N-terminal maltose-binding protein (MBP) tag followed by a TEV protease cleavage site. The overexpressed MBP–CSN8 fusion protein was initially purified by MBP affinity chromatography, followed by on-column TEV protease cleavage to remove the MBP tag. The cleaved protein was then further purified by ion-exchange chromatography. The CSN-7mer complex—which comprises all subunits except CSN5—was reconstituted by incubating the CSN1–2–3, CSN4–6–7b and CSN8 a molar ratio of 1:1.5:2. The assembled complex was then purified by ion-exchange and size-exclusion chromatography.
The CSN5 subunit was prepared from insect cells using a Bac-to-Bac expression system. Wild-type and mutant CSN5 were subcloned into a modified GTE vector (Invitrogen) containing an N-terminal octa-histidine (8×His) tag, followed by a Venus fluorescent protein and a TEV protease cleavage site. High-titre baculovirus was generated using ExpiSF9 insect cells (Thermo Fisher Scientific) cultured at 27 °C. For large-scale protein production, ExpiSF9 cells at a density of 2 × 106 cells per millilitre were infected with baculovirus and cultured at 27 °C for 68 h. Cells were harvested by centrifugation, resuspended in lysis buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM PMSF and Roche Complete Protease Inhibitor and lysed by sonication. The lysate was clarified by centrifugation and CSN5 was purified through a series of chromatography steps, including His-tag affinity, ion-exchange and size-exclusion chromatography. To obtain untagged CSN5, the Venus tag was removed by TEV protease digestion performed during dialysis following the initial His-affinity purification. The resulting mixture was then passed through a second Ni-NTA column to separate untagged CSN5 from the cleaved His–Venus tag. To assemble the complete CSN complex, purified CSN5 was incubated with tag-free CSN-7mer at a 2:1 molar ratio on ice for 30 min and then purified by size-exclusion chromatography (Superdex 200) using a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl and 0.2 mM TCEP. The peak fractions containing CSN complex were pooled, concentrated, flash-frozen and stored at –80 °C.
Preparation of N8~CRL1 protein complexes
Two short unstructured segments in the N-terminus of CUL1 (residues 1–12 and 58–81) were removed from the full-length human CUL1, resulting in CUL1ΔN (referred to here as CUL1). Both CUL1 and RBX1 (residues 16–108) were fused with an N-terminal 6xHis tag followed by a TEV cleavage site and co-expressed in BL21(DE3). The complex was initially purified using Ni2+-Sepharose affinity chromatography. Following TEV cleavage to remove the His tag, the complex was further purified by cation-exchange and size-exclusion chromatography. The same strategy was used to prepare other CRL complexes, including CUL2-RBX1, CUL3-RBX1, CUL4A-RBX1 and CUL5-RBX2.
To prepare the heterodimeric N8-activating enzyme APPBP1–UBA3, APPBP1 was subcloned into the modified pGEX4T1 vector containing a GST tag followed by a TEV protease cleavage site, whereas UBA3 was subcloned into a modified pET15b vector containing a chloramphenicol resistance cassette. GST–APPBP1 and UBA3 were co-expressed in E. coli BL21(DE3) and purified by glutathione-affinity chromatography. After TEV cleavage, the APPBP1–UBA3 complex was further purified by anion-exchange and size-exclusion chromatography. The N8-conjugating enzyme UBC12 and N8 (wild-type and mutant forms) were subcloned into the same modified pGEX4T1 vector, expressed in E. coli BL21(DE3), and purified via glutathione-affinity and anion-exchange chromatography. A truncated form of N8 ending at glycine 76, representing the mature processed form, was used throughout this study. To generate the neddylated CRL1 complex (N8~CRL1), 10 μM of purified CRL1 was incubated with 10 μM GST–N8 in the presence of 0.2 μM APPBP1–UBA3 and 0.5 μM UBC12 at 4 °C for 1 h. The neddylation reaction was conducted in buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.4 mM TCEP, 2 mM ATP and 10 mM MgCl 2 . The resulting GST–N8~CRL1 was separated from unmodified CRL1 by glutathione-affinity chromatography. After on-column TEV cleavage, N8~CRL1 was eluted from the column and further purified by cation-exchange and gel-filtration chromatography.
To conveniently monitor the production of free N8 during CSN-mediated deneddylation, N8 was site-specifically labelled with the fluorescent dye Alexa Fluor 633 (Sigma) via a cysteine residue engineered in place of the native N-terminal methionine. Excess dye was removed by size-exclusion chromatography. The fluorescently labelled N8 was then conjugated to the CUL–RBX1 complex using the neddylation protocol described above. For BLI experiments measuring the binding of N8 to CSN, an AviTag followed by a biologically inert GB1 tag (the B1 domain of streptococcal protein G; 56 residues, ~7 kDa) was fused to the N-terminus of N8. The purified Avi–GB1–N8 fusion protein was efficiently biotinylated in vitro using E. coli biotin ligase (BirA). Excess free biotin was removed by size-exclusion chromatography on a Superdex 75 column, yielding monodisperse, biotinylated Avi–GB1–N8 suitable for immobilization on streptavidin biosensors.
Biolayer interferometry
The binding affinity between N8 and CSN or CSN5–CSN6 was measured using the Octet Red 96 system (Sartorius). Streptavidin (SA) biosensors (Sartorius) coated with streptavidin were loaded with 200 nM biotinylated N8 and then quenched with 200 nM biocytin before performing binding analyses. The reactions were conducted in black 96-well plates maintained at 30 °C. The binding buffer contained 20 mM HEPES pH 7.5, 150 mM NaCl, 0.2 mM TCEP, 0.1% Tween-20 and 0.1 mg ml–1 ovalbumin. CSN or CSN5–CSN6 were tested as analytes at threefold serially diluted concentrations in the absence or presence of 20 µM CSN5i-3 (MedChemExpress). To assess the binding affinity between CSNDM and N8~CRL1 containing wild-type or 3A and 3S mutant RBX1, biotinylated N8~CRL1 was initially loaded to streptavidin-coated biosensors. CSNDM was tested as analyte at threefold serially diluted concentrations. To measure the binding affinity of CSN5i-3 with CSN or CSN5–CSN6, biotinylated anti-Venus nanobody was initially bound to streptavidin-coated biosensors to convert the probes to Venus-nanobody biosensors. Venus-tagged CSN was then loaded through the interaction between Venus and the anti-Venus nanobody. To measure the binding of CSN5i-3 to CSN5–CSN6, biotinylated CSN5–CSN6 protein was loaded to streptavidin-coated biosensors. CSN5i-3 were measured as analyte at threefold serially diluted concentrations. Data analysis of all BLI experiments was performed using Octet data analysis software, and K D was determined from either kinetics or steady-state equilibrium measurements. All BLI experiments were performed a minimum of three times.
Isothermal titration calorimetry
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