Structural basis for transthiolation intermediates in the ubiquitin pathway

Cloning

Cloning of S. pombe Uba1, Cys-Ub with an N-terminal Ulp1-cleavable His₆–Smt3 tag, and Ub without tags was described previously^15,17,52. All mutations were generated using PCR-based mutagenesis. Constructs encoding full-length S. pombe Ubc4(C21S/C107S) with a C-terminal Gly–Gly linker followed by thrombin-cleavable His₆ tag, and constructs for full-length S. pombe Ubc4 with C-terminal His₆ tag were constructed using the pET29b vector (Novagen). The DNA encoding S. pombe Pub2^HECT (amino acid residues 295–671) was inserted into the pSMT3 vector to introduce an N-terminal Ulp1-cleavable⁵³ His₆ Smt3 tag. The DNA encoding S. pombe Ub lacking the last two amino acids at its C terminus, with StrepTag and TEV protease cleavage site (MWSHPQFEKSAENLYFQGSGG) added at its N-terminus, further referred to as Ub(–2), was inserted into vector pTXB1 (New England Biolabs), to generate a C-terminal fusion with an Mxe intein-chitin binding domain.

Protein expression and purification

DNA plasmids encoding recombinant proteins were expressed in Escherichia coli strain BL21 (DE3) codon plus (Stratagene). To produce Pub2^HECT, cells were grown in Superbroth at 37 °C to OD₆₀₀ = 0.8, cooled in an ice/water bath for 20 min before addition of 1-thio-β-d-galactopyranoside (IPTG) to a final concentration of 0.4 mM. Cultures were incubated for 14–18 h at 18 °C. Uba1, Ubc4, Cys-Ub and untagged Ub were expressed as described previously^15,17,52. Cells were collected by centrifugation at 4,000g (Beckman JLA-8.1000) for 20 min at 4 °C. Cell pellets were resuspended in 50 mM sodium HEPES pH 8.0, 350 mM NaCl, 20% sucrose and snap frozen in liquid nitrogen. Cell pellets were thawed, supplemented in lysis buffer containing 0.5 mM TCEP, 2.5 mM MgCl₂, 0.1 mg ml^−1 DNAse I, 1 mg ml^−1 lysozyme, 1 mM PMSF, and lysed by sonication (2 s on, 8 s off, 50% output amplitude) twice for 3 min with a 3 min break in between using Digital Sonifier 450 Cell Disruptor (Branson) on ice/water bath. Lysates were clarified by centrifugation at 47,000g (Beckman JA-20).

For purification of His₆-tagged proteins, supernatant lysates were mixed with 15 mM imidazole and applied to Ni²⁺-NTA superflow resin (Qiagen) by gravity flow. Beads were washed with buffer containing 20 mM sodium HEPES pH 8.0, 350 mM NaCl, 0.2 mM TCEP, and 20 mM imidazole at 4 °C. Proteins were eluted in buffer containing 20 mM sodium HEPES pH 8.0, 350 mM NaCl, 0.2 mM TCEP, and 250 mM imidazole at 4 °C. His₆–Smt3 tags used for affinity purification were removed by incubation with Ulp1⁵³ and separated by size-exclusion chromatography (SEC) with Pub2 and Ubc4 separated using HiLoad 26/600 Superdex 75 PG column and Uba1 separated using HiLoad 26/600 Superdex 200 PG column (GE Healthcare), equilibrated in 20 mM sodium HEPES pH 7.5, 250 mM NaCl, 0.1 mM TCEP at 4 °C. For Ubc4(C21S/C107S), the C-terminal His₆ tag was removed by treatment with thrombin then separated by SEC. After SEC, Uba1 was further purified by anion-exchange chromatography using a MonoQ 10/100 GL column (GE Healthcare) in 20 mM sodium HEPES pH 8.0, 0.1 mM TCEP and a linear gradient of 50–800 mM NaCl at 4 °C. To obtain fluorescein-Ub variants, Cys-Ub variants containing an additional N-terminal Cys for labelling were obtained as described previously¹⁵. Purified Cys-Ub variants were incubated in 20 mM HEPES pH 7.5, 200 mM NaCl, 2 mM TCEP for 10 min at room temperature, desalted into the same buffer without TCEP and modified by adding 10-fold molar excess of 5-fluorescein maleimide (Thermo Fisher Scientific) and incubating for 2 h at room temperature. Reactions were then buffer exchanged to 20 mM sodium HEPES pH 7.5, 200 mM NaCl using 7 kDa MWCO Zeba Spin Column (Thermo Fisher Scientific) and separated by size exclusion using Superdex 75 Increase 10/300 GL column (GE Healthcare) equilibrated in the same buffer at 4 °C. Native Ub (without affinity tags), was purified as described previously⁵², with additional SEC using a HiLoad 26/600 Superdex 75 prep grade column (GE Healthcare) equilibrated in 20 mM Tris•HCl pH 7.5, 250 mM NaCl at 4 °C. Fractions containing the desired proteins were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C.

Preparation of Ub-PSAN

H₂N-Gly–PSAN was synthesized as described²⁸. Ub^1–74 fused to Mxe intein-chitin binding domain was expressed, and cells collected and lysed using the same procedure as for Pub2^HECT. Lysate supernatant was incubated with chitin beads (New England BioLabs) equilibrated in 30 mM sodium HEPES pH 8.0, 350 mM NaCl. Beads were washed with ten bed volumes of equilibration buffer and then two bed volumes of 30 mM Bis-Tris•HCl pH 6.5, 350 mM NaCl at 4 °C. The Ub^1–74~2-mercaptoethanesulfonate (MESNa) thioester was obtained by incubating the resin in 2 bed volumes of 30 mM Bis-Tris•HCl pH 6.5, 350 mM NaCl, 200 mM MESNa at room temperature for 12–16 h. The cleaved protein was eluted and transferred to ice/water bath, then the resin was treated again with cleavage buffer for 12 h at 4 °C, followed by elution. Eluted fractions were combined, concentrated using 3 kDa MWCO Amicon filter (Milipore) to 8–10 mg ml^−1 and treated with 1 M hydrazine at 30 °C for 30 min. The resulting Ub^1–74 hydrazide was separated by SEC using HiLoad 26/600 Superdex 75 prep grade column (GE Healthcare) equilibrated with 25 mM sodium phosphate pH 6.5, 350 mM NaCl at 4 °C. Fractions containing Ub^1–74 hydrazide were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C.

To generate Ub^1–74 acyl azide for coupling with H₂N-Gly–PSAN, Ub^1–74 hydrazide, at a concentration of 3 mM, was combined with 100 mM sodium citrate pH 3.0 on ice/water bath. NaNO₂ was added to a final concentration of 50 mM (from 0.5 M stock adjusted to pH 5 with HCl on ice/water bath immediately before use) and the reaction transferred to −20 °C (ice:NaCl bath at 3:1 ratio) for 5 min. An equal volume of 250 mM H₂N-Gly–PSAN (HCl salt) in 1 M sodium HEPES pH 8 was added immediately after dissolving and the reaction incubated for 10 min on ice/water bath followed by 10 min at room temperature, and buffer exchanged into 20 mM sodium HEPES pH 8, 350 mM NaCl at 4 °C using a 7 kDa MWCO Zeba Spin Column (Thermo Fisher Scientific). Ub-PSAN was separated by SEC using Superdex 75 Increase 10/300 GL column (GE Healthcare) equlibrated in 20 mM sodium HEPES pH 8, 350 mM NaCl at 4 °C. The molecular weight of Ub-PSAN was confirmed by ultraperformance liquid chromatography electrospray mass spectrometry using Acquity UPLC-MS (Waters). Ub-PSAN was concentrated to ~1 mM, flash-frozen in liquid nitrogen, and stored at −80 °C.

Preparation of E1–Ub(T)–E2 and E2–Ub(T)–E3^HECT complexes

To obtain E2–Ub vinyl thioether for reactions with E1 and E3^HECT, a mixture of 0.6 mM Ub-PSAN, 0.4 mM Ubc4(C21S/C107S), 20 mM sodium HEPES pH 8.0, 200 mM NaCl was incubated for 1 h at 4 °C. The E2–Ub vinyl thioether product was separated by SEC on a HiLoad 26/600 Superdex 75 prep grade column (GE Healthcare) equilibrated in the same buffer.

To obtain E1–Ub(T)–E2, a mixture of 75 μM E2–Ub vinyl thioether, 50 μM Uba1 (lacking the first 12 amino acids), 20 mM sodium HEPES pH 8.0, 100 mM NaCl, 0.2 mM TCEP was incubated for 1 h at room temperature, and then incubated on ice/water bath. The 30 ml total reaction volume was divided into four 7.5 ml portions. Each portion was applied to a 5 ml StrepTrap HP column (GE Healthcare) equilibrated in Tris•HCl pH 7.2, 200 mM NaCl, 0.2 mM TCEP at 4 °C. After washing with 10 column volumes of equilibration buffer, proteins were eluted in the same buffer containing desthiobiotin at a final concentration of 2.5 mM. Eluted fractions from all runs were combined and incubated with TEV protease for 9 h at 4 °C to remove the StrepTag–TEV tag from Ub. After tag cleavage, E1–Ub(T)–E2 was separated by anion-exchange chromatography using MonoQ 5/50 GL column (GE Healthcare) in 20 mM Tris•HCl pH 7.2, 0.1 mM TCEP and a linear gradient of 50–400 mM NaCl at 4 °C. Fractions containing E1–Ub(T)–E2 were concentrated to ~12 mg ml^−1, flash-frozen in liquid nitrogen, and stored at −80 °C.

To obtain E2–Ub(T)–E3^HECT, a 30 ml solution containing 75 μM E2–Ub vinyl thioether, 75 μM Pub2^HECT, 20 mM sodium HEPES pH 8.0, 200 mM NaCl, 0.5 mM TCEP was incubated for 8 h at room temperature, then moved to ice/water bath. The reaction mixture was subjected to StrepTag affinity chromatography and TEV protease cleavage as described above for E1–Ub(T)–E2 complex. After cleavage, E2–Ub(T)-E3^HECT was isolated with two successive rounds of anion-exchange chromatography using a MonoQ 5/50 GL column (GE Healthcare) equilibrated with 20 mM Tris•HCl pH 7.5, 0.1 mM TCEP with a linear gradient of 50–280 mM NaCl at 4 °C. Fractions containing E2–Ub(T)–E3^HECT were concentrated to ~13.5 mg ml^−1, flash-frozen, and stored at −80 °C.

Cryo-EM sample preparation and data collection

Prior to grid preparation, an aliquot of E1–Ub(T)–E2 or E2–Ub(T)–E3^HECT was rapidly thawed in a room temperature water bath and centrifuged for 10 min at 4 °C at 18,000g. E1–Ub(T)-E2 was diluted to 3 mg ml^−1 in Tris•HCl pH 7.2, 100 mM NaCl and preincubated for 30 min on ice/water bath with 5 mM MgCl₂, 1 mM ATP, and ~0.8 molar equivalents of Ub. E2–Ub(T)–E3^HECT was diluted to 4.5 mg ml^−1 in Tris•HCl pH 7.2, 100 mM NaCl. Prior to vitrification, CHAPSO was added to samples at final concentrations of 0.05% and 0.1% w/v for E1–Ub(T)–E2 and E2–Ub(T)–E3^HECT complexes, respectively. Four microlitres of sample was applied to freshly glow-discharged UltrAuFoil 300 mesh R1.2/1.3 grids (Quantifoil) at 100% humidity at 25 °C. After 8 s, samples were blotted for 3.5–4.0 s and vitrified by plunging into liquid ethane using a Vitrobot Mark IV (FEI-Thermo Fisher).

Cryo-EM data were collected at MSK Richard Rifkind Center for Cryo-EM, using a Titan Krios transmission electron microscope (FEI-Thermo Fisher) operated at 300 keV. Cryo-EM movies (40 frames per movie, 4 s exposure time) were recorded at a dose rate of ~20 e^− pixel^−1 s^−1 using a K3 Summit direct electron detector (Gatan) operated in super-resolution mode at a physical pixel size of 1.064 Å. Automated data collection was performed in Serial EM⁵⁴ using image shift to record data from nine ice holes per stage movement. Two datasets for E1–Ub(T)–E2 were obtained with 12,132 and 14,545 movies each, and 2 datasets for E2–Ub(T)–E3^HECT were obtained with 15,705 and 14,101 movies each.

Cryo-EM image processing

Initial data processing steps were similar for E1–Ub(T)–E2 and E2–Ub(T)–E3^HECT. Movie frames from each session were gain normalized, 2× Fourier cropped, aligned and summed with and without dose-weighting using MotionCor2⁵⁵. Estimation of the contrast transfer function (CTF) was performed using Gctf⁵⁶ from non-dose weighted micrographs. Micrographs with estimated resolution limits worse than 4.5 Å, poor CTF fit or with crystalline ice were discarded. Initial particle sets were obtained by reference-free auto-picking with Laplacian-of-Gaussian filtering in RELION 3.1^57,58. Subsequent steps were performed in cryoSPARC 4.0.2⁵⁹ with the exception of particle picking using Topaz⁶⁰ and Bayesian polishing performed in RELION 3.1⁶¹. UCSF Pyem was used to convert particle metadata from cryoSPARC to RELION format⁶². Particles from each dataset underwent several rounds of 2D classification to remove junk particles and to obtain subsets from 2,000 random micrographs for training neural network-based particle picking in Topaz. Trained models were applied to full datasets, and identified particles were extracted using a 256-pixel box size.

For E1–Ub(T)–E2, Topaz-picked particles were used in rounds of 2D classifications to remove junk particles. Two successive rounds of ab initio 3D reconstruction and heterogeneous refinement were performed, first with three classes, then with four, each time removing classes lacking secondary structure features. 1,826,497 particles were re-extracted using a 384-pixel box size and pooled for a single ab initio 3D reconstruction followed by non-uniform 3D refinement⁶³, resulting in a consensus map with a nominal resolution of 3.0 Å. To remove low-quality particles, four consecutive rounds of heterogeneous refinement were initialized with four copies of a consensus map lowpass filtered to 15 Å and one copy lowpass filtered to 30 Å. Particles assigned to the lowest resolution class were discarded after each round. Particles were recentred and subjected to non-uniform 3D refinement and Bayesian polishing, followed by 2D classification to remove images with artefacts resulting in 1,610,345 particles that yielded a consensus map with a nominal resolution of 2.64 Å, which improved to 2.51 Å after per-particle defocus refinement, per optics group estimation of the beam tilt, trefoil, spherical and fourth-order aberrations, followed by second round of per-particle defocus refinement. Particles were next subjected to heterogeneous refinement (with 6 classes and 2× binning) initialized with 20 Å lowpass-filtered consensus map resulting in four classes with Ub(A) and two without Ub(A) that were combined to yield two particle sets for doubly and singly loaded complexes (1,295,595 and 314,750 particles respectively), which after non-uniform 3D refinement, resulted in consensus maps at nominal resolutions of 2.50 and 2.79 Å, respectively. To resolve Ub(T) conformations in doubly and singly loaded complexes, particles were then subjected to 3D classification without image realignment (10 classes, target resolution 7 Å, number of O-EM epochs 8, O-EM learning rate init 0.3, initialization mode principal components analysis (PCA), force hard classification on) with a mask focused on Ub(T) region. Maps were generated by 3D reconstruction using particles from each class and their alignment information and a half-set splits from the gold-standard refinement of their parental sets (consensus maps). The 3D classes used for model building were further subjected to non-uniform 3D refinement. The continuum of E1 SCCH rotation relative to E1 adenylation domains (IAD and AAD) was resolved by 3D variability analyses⁶⁴ (3 modes to solve, filter resolution 5 Å) performed separately for particles corresponding to doubly and singly loaded complexes, using a mask encompassing the E1–E2 region but excluding Ub(T) regions. In both cases, particles were sorted into five clusters based on values of the latent coordinate of the component 1, best capturing rotation of E1 SCCH domain. Five clusters were chosen because this number adequately tracked SCCH movement (1.5° to 2° of rotation per cluster) while ensuring sufficient particles within each cluster for further analysis. Particles from each cluster were subjected to non-uniform refinement, resulting in maps with nominal resolutions of 2.95–3.16 Å for singly loaded complexes and 2.67–2.86 Å for doubly loaded complexes. Ub(T) conformations in each cluster were resolved through 3D classifications without image realignment (10 classes, target resolution 7 Å, number of O-EM epochs 8, O-EM learning rate init 0.3, initialization mode PCA, force hard classification on) using a focus mask on the Ub(T) region. Maps were generated by 3D reconstruction using particles from each class and their alignment information and half-set splits from the gold-standard refinement of parental sets (clusters 1–5). The 3D classes used for model building were further subjected to non-uniform 3D refinement. Where applicable, maps were lowpass filtered to 5 Å using a 10th-order Butterworth filter. Statistics for data collection are listed in Supplementary Table 1.

For E2–Ub(T)–E3^HECT, Topaz-picked particles were selected after 2D classification from each dataset and re-extracted using 320-pixel box size, combined, and subjected to rounds of 2D classification, resulting in 2,704,150 particles. Ab initio 3D reconstructions using six classes were performed, followed by heterogeneous refinement (with 2× binning). Particles from classes with defined secondary structure features (2,511,724 particles) were combined and used in single ab initio 3D reconstruction followed by non-uniform 3D refinement to a map with a nominal resolution of 3.30 Å. Particles were recentered, subjected to non-uniform 3D refinement and Bayesian polishing, followed by 2D classification to remove images with artefacts, and per-particle defocus refinement, resulting in 2,428,313 particles that yielded a consensus map with a nominal resolution of 3.09 Å. Subsequently, 3D variability analysis (3 modes to solve, filter resolution 5 Å) was performed with a mask encompassing the entire complex. Particles were then split into 20 clusters based on all solved modes of 3D variability, an empirically determined number that resulted in best separation between complexes. Each cluster was subjected to non-uniform 3D refinement, resulting in maps with nominal resolutions of 3.27–3.56 Å. The 8 clusters resulted in maps with no apparent or poorly resolved Ub(T) density. Ub(T) densities were resolved in maps for 12 clusters with 6 maps capturing Ub(T) at positions proximal to E2 and 6 other maps with Ub(T) in positions more distal from E2 (states 2–7). All 12 maps revealed Ub(T) C-terminal residues between E2 and E3^HECT. Particles from 6 clusters containing Ub(T) at a position most proximal to E2 (790,208 particles) were combined and used for non-uniform 3D refinement resulting in a map with a nominal resolution of 3.17 Å. To select the best particles and to improve the map at the transthiolation active site, a 3D classification without image alignment (4 classes, target resolution 7 Å, number of O-EM epochs 8, O-EM learning rate init 0.3, initialization mode PCA, force hard classification on) was performed with a mask focused on the transthiolation site. One of four classes showed improved density corresponding to the transthiolation site, including E2 amino acids around its active site. This class containing 204,763 particles was subjected to non-uniform 3D refinement to yield a map with a nominal resolution of 3.23 Å (state 1). Reported resolutions were determined using the gold-standard 0.143 criterion based on Fourier shell correlation. Statistics for data collection are listed in Supplementary Table 1.

Model building and refinement

Initial coordinates were generated by docking individual chains from reference structures into cryo-EM maps in UCSF Chimera⁶⁵ followed by manual building in Coot⁶⁶. For E1–Ub(T)–E2, the crystal structures of Uba1–Ubc4/Ub/ATP·Mg and Ub (Protein Data Bank (PDB): 4II2¹⁷ and 6O82¹⁵, respectively) were used. For E2–Ub(T)–E3^HECT, a homology model of Pub2^HECT was obtained from SWISS-MODEL⁶⁷ based on crystal structures of UbcH5B∼Ub-NEDD4L (PDB: 3JW0²⁵), Ubc4 and Ub (PDB: 4II2¹⁷ and 6O82¹⁵, respectively). Coordinates for all models were produced via iterative rounds of refinement and building in real space using Phenix and Coot^66,68. Geometry restraints for the linker (transthiolation intermediate analogue) were generated using Phenix.elbow⁶⁹. MolProbity was used to evaluate model integrity⁷⁰. Structure representations were generated using UCSF ChimeraX⁷¹. 2D slice views of electron microscopy maps were visualized using IMOD 4.11⁷². Statistics for model refinement are listed in Supplementary Table 1.

Preparation of singly loaded fluorescein-Ub~E1 complex

To generate thioester-linked fluorescein-Ub~E1 complex, 125 μl reaction containing full-length Uba1 (8 μM), substochiometric amount of fluorescein-Ub (6 μM) in 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 0.1 mM TCEP, 5 mM MgCl₂ and 1 mM ATP was incubated at 10 min at 30 °C. The mixture was then cooled to 4 °C and separated by anion-exchange chromatography using MonoQ 5/50 GL column (GE Healthcare) in 20 sodium HEPES pH 7.5, 0.1 mM TCEP and a linear gradient of 100–400 mM NaCl. SDS–PAGE analysis of purified fluorescein-Ub~E1 incubated with and without β-mercaptoethanol confirmed its sensitivity to reducing agent, consistent with a singly loaded fluorescein-Ub~E1 and without Ub bound non-covalently to the adenylation site.

E1–E2 single-turnover Ub thioester transfer assays

The Ub~AMP mimic (Ub-AMSN) was synthesized as described previously^14,15,34. Single turnover assays were performed on ice/water bath. For each replicate, singly loaded fluorescein-Ub~E1 complex (10 nM) was freshly prepared and preincubated in 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 0.1 mM TCEP, 0.1 mg ml^−1 ovalbumin, either alone or with addition of ATP (4 mM), Ub and ATP (10 μM and 4 mM, respectively), Ub-AMSN (10 μM), or Ub-AMSN and PP_i (10 μM and 4 mM, respectively) in 400 μl for 5 min. A control without addition of Ubc4 (E2) was obtained by diluting a 100 μl aliquot with an equal volume of 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1 mM TCEP, 0.1 mg ml^−1 ovalbumin. Thioester transfer (chase) was initiated by diluting a 100 μl aliquot with an equal volume of 100 nM E2, 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1 mM TCEP, 0.1 mg ml^−1 ovalbumin. The resulting concentrations at time 0 were 5 nM for fluorescein-Ub~E1 complex and 50 nM for E2. Aliquots from indicated timepoints and a control without E2 were quenched by addition of LDS NuPAGE buffer supplemented with EDTA (final concentrations of 1× and 50 mM, respectively). Products were separated on 4–12% NuPAGE BIS-Tris gels with 1× MOPS running buffer (Thermo Fisher Scientific). To increase fluorescence signal by converting fluorescein to its di-anionic form, gels were incubated in 50 mM Tris•HCl pH 9.5 for 3 min prior to scanning using Amersham Typhoon 5 (Cytiva). Bands were quantified using ImageQuant software (Cytiva).

E1–E2 multiple turnover Ub thioester transfer assays

E1–E2 Ub thioester transfer assays were performed in 160 μl reactions with 1.5 nM full-length Uba1, 200 nM of indicated variant of Ubc4, 5 μM Ub, 20 mM sodium HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 0.1 mM TCEP. Reactions were conducted at 22 °C and initiated by adding ATP to a final concentration of 200 μM including a control that lacked ATP. Aliquots were removed at indicated timepoints and quenched by addition of LDS NuPAGE buffer supplemented with EDTA (final concentrations of 1× and 50 mM, respectively) and products were separated on 12% NuPAGE BIS-Tris gels with MOPS running buffer (Thermo Fisher Scientific). Gels were stained using Flamingo dye (Bio-Rad), scanned using Amersham Typhoon FLA 9500 and quantified using ImageQuant software (Cytiva).

E2~Ub(T) to E3^HECT pulse–chase Ub thioester transfer assays

For E2~Ub(T) to E3^HECT~Ub thioester transfer assays, for each replicate, indicated mutational variants or wild-type Ubc4 (7 μM) were charged with wild type or indicated mutational variant of fluorescein-Ub (12 μM) using E1 (300 nM) in 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 2 mM ATP, 0.1 mM TCEP in 40 μl reaction volume. After incubation for 30 min at 25 °C, reactions were transferred to ice/water bath and E1 activity was quenched by fourfold dilution with 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 40 mM EDTA. Subsequent steps were performed on ice/water bath. Mixtures were diluted with 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 0.1 mM TCEP, 0.1 mg ml^−1 ovalbumin to a final E2~Ub(T) concentration of 30 nM. A control without E3^HECT was obtained by diluting a 100 μl aliquot with an equal volume of 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 0.1 mM TCEP, and 0.1 mg ml^−1 ovalbumin. Thioester transfer reactions (chase) were initiated by diluting a 100 μl aliquot with an equal volume of a solution of a specific variant of Pub2^HECT at 40 nM in 20 mM sodium HEPES pH 7.5, 100 mM NaCl, 0.1 mM TCEP, and 0.1 mg ml^−1 ovalbumin. Concentrations at time 0 were 15 nM for fluorescein-Ub~E2 and 20 nM for E3^HECT. Aliquots (40 μl) were removed at indicated timepoints and quenched by addition of LDS NuPAGE buffer (final concentration of 1×). Products were separated on 4–12% NuPAGE BIS-Tris gels with MOPS running buffer (Thermo Fisher Scientific). To increase fluorescence signal by converting fluorescein to its di-anionic form, gels were incubated in 50 mM Tris•HCl pH 9.5 for 3 min prior to scanning using Amersham Typhoon 5 (Cytiva). Bands were quantified using ImageQuant software (Cytiva).

Statistics and reproducibility

Generation of the E1–Ub(T)–E2 and E2–Ub(T)–E3 transthiolation analogues for structural analysis was reproduced with at least three independent purifications. All biochemical experiments were replicated three times, reproduced with at least two independent purifications, and reproduced independently at least twice. Statistical analyses and graphing of the data were performed using Prism 10.1.0 (GraphPad Software). Number of replicates and details on statistical analyses and test are provided in the methods pertaining to each experiment and/or the appropriate legend.

Reporting summary

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