Structure and mechanism of the HECT ligase HECTD3

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: minor_revision

Abstract
HECTE3ligasesregulatemanycellularprocesses,yethowtheyrecognisetheir substratesandsynthesisespecifictypesofpoly-ubiquitinchainsisstill incompletelyunderstood.HECTD3,amemberofthe“otherHECT”family,is implicatedintheregulationofinflammation,apoptosis,andinfectionand highlyexpressedinseveralcancers.Thesefunctionsarelargelyattributedto itsligaseactivityandmodificationofdiversesubstrateswithdifferenttypesof ubiquitinchains.Wepresentadetailedanalysisoftheligaseactivityof HECTD3,includingitsubiquitinlinkagepreferences,oligomericstateand substrateubiquitination.Usingcryo-EM,weprovidethefull-lengthstructures ofHECTD3inbothapoandubiquitin-loadedforms,revealingkeyinsightsinto itsdomainorganisation,includingdiscoveryofadistinctfoldoftheN-terminal region,andmechanisticfeatures.SomeofthesearesharedwithotherHECT ligases,whileothersareuniquetoHECTD3andcontributetodifferencesinits catalyticmechanismsandfunctionaldiversity.

Introduction
The covalent attachment of ubiquitin to proteins, lipids, or sugars regulates diverse cellular processes1–4. The type of ubiquitin modification influences substrate stability, localisation, interactions, or activity5. Modification of substrates with poly-ubiquitin chains dictates distinct cellular outcomes depending on chain architecture; for instance, K48 chains typically signal proteasomal degradation, while K63-linked chains regulate signal transduction and DNA damage responses6. Ubiquitin attachment occurs through a three-enzyme cascade of E1-activating enzymes, E2-conjugating enzymes, and E3 ligases. Humans encode over 600 E3 ligases that function via distinct mechanisms: RING ligases transfer ubiquitin directly from the E2 to the substrate, while RBR and HECT ligases form an intermediate thioester bond with ubiquitin and determine chain type specificity7,8. The human HECT ligases vary in molecular weight from 80.5 kDa of the smallest (G2E3) to over 500 kDa for the largest member (HERC2). They all contain a C-terminal catalytic HECT domain, but differ in the composition of their N-terminal substrate binding and regulatory domains, which are used to categorise them into three subfamilies: NEDD4-type HECT ligases harbour N-terminal C2 and WW domains, HERC family ligases contain RCC1 domains, and the remainder form the “other HECT” family with diverse N-terminal composition9. The ~45 kDa catalytic HECT domain has a bilobal structure comprising an E2 binding N-lobe, and a catalytic cysteine-containing C-lobe9. A short intervening linker allows conformational flexibility that is essential for catalysis10. During the catalytic cycle, the lobes adopt two distinct conformations that differ in their relative orientation: the E3 receives the ubiquitin (UbD) from the E2 in the “inverted T-shape” before transferring ubiquitin to the substrate in the L-conformation11–14. While all HECT ligases share the catalytic HECT domain, the extent to which their regulatory mechanisms and substrate recognition strategies are conserved, is only beginning to emerge. A number of cryo-EM structures of large full-length HECT ligases showed that many adopt ring-shaped structures and α-solenoid helical scaffolds, and that some self-associate into higher order structures15–19. The activity of some HECT ligases has been shown to be regulated by autoinhibitory interactions20,21, phosphorylation or membrane interaction22, however, these regulatory mechanisms are often protein specific without a general underlying theme.

HECTD3 is a member of the “other HECT” family of modest size (97 kDa) which has been associated with several biological processes, including inflammation, apoptosis and viral and bacterial infection23–26. It is highly expressed in many cancers, including glioma, breast, gastric and ovarian cancer, and has been shown to play a role in metastasis and drug resistance27–29. Most functions have been linked to its ubiquitin ligase activity and modification of different substrates with varying types of ubiquitin chains. For example, HECTD3 was shown to ubiquitinate Caspase-9 to promote oesophageal squamous cell carcinoma (ESCC) growth and cell survival30. K63-linked ubiquitination of Caspase-8 was shown to promote breast cancer cell survival31 and of poly-ADP-ribosyltransferase PARP1 to promote glioblastoma tumorigenesis32. Modification of c-MYC with K29 linked ubiquitin chains has been reported to promote progression in gastric cancer cells33, while ubiquitination of STAT3 has been associated with multiple sclerosis34. Despite its important physiological functions and therapeutic potential, substrate selection, chain linkage specificity and regulatory mechanisms of HECTD3 remain poorly understood. Furthermore, it is unknown whether it shares features observed in other HECT ligases, such as oligomerisation or formation of ring-shaped structures, especially given that its N-terminal region has no recognisable fold.

Here, we provide an in-depth functional and structural analysis of the ubiquitination activity of HECTD3. We provide insights into E2 preference and linkage selectivity, as well as the oligomeric state of HECTD3. Using cryo-EM, we provide the full-length structures of HECTD3 in both apo and ubiquitin-loaded forms, revealing key details of its domain organisation and mechanistic features. Some of these are shared with other HECT ligases, while others are unique to HECTD3, offering new perspectives on its catalytic mechanisms and functional diversity.

Results
HECTD3 is a constitutively active E3 with unique E2 preference
HETCD3 is highly conserved across vertebrates (Supplementary Fig. 1). We first assessed if catalytic activity of HECTD3 might be regulated through autoinhibitory interactions by comparing activities of full-length HECTD3 with that of two HECT domain constructs of differing lengths (Fig. 1a–c, Supplementary Fig. 2a–c). Ubiquitination assays were carried out using members of the promiscuous UBE2D E2 family and the cysteine-specific E2 UBE2L3. The autoubiquitination activity observed provided no evidence for autoinhibitory interactions and even suggested that the full-length protein might be more active. Surprisingly however, whereas we detect activity with all UBE2D family members, none is observed with UBE2L3 which is active with many other HECT E3s and often assumed to be the cognate E2 for HECT ligases (Fig. 1b, c). In line with this observation, we detect only minimal HECTD3~Ub thioester intermediate formation with UBE2L3 (Supplementary Fig. 2d). To further investigate this observation, we carried out a screen of all E2s with western blot analysis. With this more sensitive detection, HECTD3 shows low activity with multiple different E2s including UBE2L3 but is most active with UBE2D family members (Supplementary Fig. 2e). To investigate if the apparent increased activity of full-length HECTD3 is due to more efficient charging with ubiquitin compared to the isolated HECT domain, we analysed the reaction with a ubiquitin reactive probe (Ub-propargylamide; Ub-PA) that creates a stable bond with the active site cysteine that mimics the thioester. Whereas the probe fully labels the full-length protein, only half of the HECT domain becomes modified (Fig. 1d). Similarly, when we analysed trans-thiolation from E2 to E3 we found that the full-length protein shows faster and increased loading suggesting that regions outside the core HECT domain support E3~Ub formation (Supplementary Fig. 2d).

Self-association has been reported to regulate the activity of some HECT ligases35–37. To assess the oligomeric state of HECTD3, we used SEC-MALLS which indicated that the HECT domain exists in a monomer-dimer equilibrium, whereas the full-length protein is mainly monomeric though has some tendency for self-aggregation (Fig. 1e). Nevertheless, for both proteins, the predominant format at assay conditions in solution is monomeric.

Taken together, we show that HECTD3 is a constitutively active E3 that is not regulated by intramolecular inhibition, nor self-association as has been seen in other HECT ligases. Both, the HECT domain and the full-length protein share the same E2 profile and display a clear preference for the UBE2D family and only minimal activity with UBE2L3.

Structure of apo and ubiquitin-loaded HECTD3
To gain a molecular understanding of the mechanism of HECTD3, we determined the cryo-EM structure of the protein in its apo and ubiquitin-conjugated (HECTD3-Ub) forms (Supplementary Table 1). In the apo form, HECTD3 exhibits conformational heterogeneity, with ab initio 3D reconstruction, followed by heterogeneous refinements performed in cryoSPARC, yielding two distinct conformational states (Fig. 2a, b). Despite the limited resolution of both reconstructed cryo-EM maps (~6Å, Supplementary Fig. 3a and 4), we unambiguously positioned the different HECTD3 structural elements using the higher resolution HECTD3-Ub structure as a reference model (see below). Apo HECTD3 adopts a globular structure and is present as a monomer that does not adopt a ring or solenoid shape as observed in larger HECTs (Fig. 2a). The previously uncharacterised N-terminal region is well structured and connected to the DOC domain via two flexible linkers, spatially bridging it to the the HECT domain that displays the classic bilobal structure observed in other HECT ligases. The two distinct apo-HECTD3 structures differ by the relative orientation of the HECT C-lobe and N-lobe, representing the L- and inverted T-conformation canonical of HECT ligases (Fig. 2a, b). In the L-conformation, the C-lobe is rotated 180° and translated 30Å relative to the inverted T-conformation (Supplementary Movie 1, Supplementary Fig. 3b). Each conformation is stabilised by specific interactions. In the T-state, a salt bridge between D584 of the N-lobe and R795 of the C-lobe contributes to the stabilisation (Fig. 2c). Both residues are poorly conserved in other HECT ligases but present in HECTD3’s closest relatives HERC1 and 2. Additional inter-lobe interactions occur between S796 and R583. While R583 is not a conserved residue, S796 is present in several other HECTs and might play a similar stabilising role for other family members. Besides these inter-lobe interactions, the inverted T-shape is stabilised by interactions between C-lobe Y847 and R392 of the N-terminal region. Y847 is conserved in some HECTs, including its closest relatives HERC1 and HERC2, and might be involved in interactions between the C-lobe and N-terminal domains in other HECTs as well. This interaction is not possible in the HECT domain construct and might explain the difference observed in ubiquitin loading between the HECT domain and the full-length protein.

The L-conformation of apo-HECTD3 represents the state adopted by HECT domains following ubiquitin loading when it is poised for transfer to the substrate. To characterise the ubiquitin-loaded form of HECTD3, we used the ubiquitin reactive probe Ub-PA (ubiquitin-propargylamide) to generate a stable E3-Ub adduct. Loading with ubiquitin resulted in a significant reduction of HECTD3 intra-domain flexibility, allowing single-particle cryo-EM reconstruction of HECTD3-Ub with a substantial improvement in the overall resolution of the final map to 3Å (Fig. 3a, b and Supplementary Fig. 5a–d and Supplementary Fig. 6). The AlphaFold model of HECTD3-Ub was fitted to the consensus map, and cycles of chain rebuilding followed by real-space refinement yielded the final structural coordinates. The DOC domain, inserted between two long loops, exhibited lower resolution compared to the rest of the protein. Therefore, we determined its high-resolution structure using X-ray crystallography (Supplementary Fig. 5e, f and Supplementary Table 2), followed by rigid body fitting of the domain into the consensus map. Ubiquitin loading fixes the HECT C-lobe in the L-conformation, that aligns well with structures of other HECTs conjugated with ubiquitin (Supplementary Fig. 5g)11–14,38. The HECTD3 C-terminal tail (residues 850-861), which is invisible in the apo structure, is structured in the HECTD3-Ub map with only the last two C-terminal residues missing.

The structure of the N-terminal region (aa 1–450) of HECTD3 is unique to this protein. The first nine N-terminal residues and two long loops (78–90 and 158–172) are invisible in the map, likely due to intrinsic flexibility. Beyond the DOC domain spanning amino acids 238–376, this region of HECTD3 is highly structured and can be divided into two subdomains: the first formed by the first two N-terminal α-helices, as well as regions spanning residues 177–230 and 381–448, consisting of nine α-helices. It provides a helical platform for the DOC domain that is connected via two flexible linkers. A second N-terminal subdomain, spanning residues 45-156, consists of seven ß-strands and one α-helix (Fig. 3c). No homologous structures of HECTD3 N-terminal domain were identified using DALI39. Low similarity was found with the putative phage lysine (DALI Z-score 3.9) and with residues 2488–2552 of the ubiquitin ligase MYCBP2 (DALI Z-score 6.3), a region that remains structurally and functionally uncharacterised. These unique domain features of HECTD3 will be referred to as D3NA and D3NB (HECTD3 N-terminal domain A and B). The D3NA domain exhibits lower overall resolution compared to the HECT domain (Supplementary Fig. 5a) which we attribute to the dynamic properties of this protein segment that harbours the substrate binding DOC domain. 3D variability analysis40 (Supplementary Movies 2–4) highlights movements of the N-terminal part of HECTD3 coupled with those of the DOC domain, which we hypothesise would be greatly reduced upon substrate binding.

To validate the single-particle cryo-EM derived structures, we recorded size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) data for both the apo and ubiquitin-loaded HECTD3 (Supplementary Table 3). SEC-SAXS analysis reveals that HECTD3-Ub is a single species in solution, whereas the apo structure exists in equilibrium between monomeric and larger species (Supplementary Fig. 7a). The molecular weight of HECTD3-Ub evaluated from the SAXS data matches the value obtained from the protein primary sequence, confirming that HECTD3-Ub is monomeric in solution. Analysis of the scattering intensities for HECTD3-Ub produces values for the radius of gyration (Rg) and particle maximum dimension that are larger than to those calculated from the HECTD3-Ub structure coordinates (Supplementary Table 3). HECTD3-Ub has an overall slightly elongated shape (Dmax > Rg) with some degree of flexibility, as evidenced by the upward trend at high q*Rg values in the dimensionless Kratky plot (Supplementary Fig. 7b). Using HECTD3-Ub cryo-EM structure coordinates to fit the experimental scattering intensities results in a χ2 value of 3.8. We conclude that HECTD3-Ub in solution likely resembles the cryo-EM structure, with discrepancies between the model and the observed X-ray scattering intensities arising from the E3 inherent interdomain flexibility and unmodelled residues in the N-terminal and long loop regions.

The structure of the HECTD3-Ub intermediate reveals unique interfaces
The high resolution of the HECTD3-Ub structure allowed a detailed analysis of the multiple interfaces between ubiquitin and the N- and C-lobes of HECTD3 (Fig. 4). Hydrophobic interactions between ubiquitin (I36, L71 and L73) and residues of the C-lobe stabilise the complex and are supported by several polar sidechain interactions, including between N848 of HECTD3 and K11 of ubiquitin (Fig. 4a, panel 1). The first helix of the HECT domain N-lobe is involved in another hydrophobic interaction interface with K6, L8 and H68 of ubiquitin (Fig. 4a, panel 2). Interestingly, we detected a salt bridge between R74 of ubiquitin’s C-terminal tail and D546 of the N-lobe. Most HECT domains have an acidic residue at this position, and a similar interaction has been observed in other HECT-Ub structures13,14,41. Mutation of HECTD3 D546 to alanine had almost no effect on the trans-thiolation activity but lead to a reduction in auto-ubiquitination activity (Fig. 4b, c) indicating that it is not essential for ubiquitin binding, but rather optimal orientation and stabilisation of the donor ubiquitin prior to substrate transfer. Additional polar interactions are formed between C-terminal residues of HECTD3 and ubiquitin: T854 of HECTD3 hydrogen bonds with the backbone ubiquitin’s C-terminal residue G75, while D853 of HECTD3 interacts with both ubiquitin R42 and R72 (Fig. 4a, panel 3). These latter interactions are crucial for ubiquitin recognition, as mutations result in significantly reduced trans-thiolation, and consequently decreased auto-ubiquitination activity (Fig. 4b, c).

The HECTD3 active site comprises the active site loop, the C-terminal tail of ubiquitin, and residues of the N-lobe (Fig. 4d). Like other HECT-ubiquitin structures, the C-terminal tail of ubiquitin is involved in hydrogen bonding networks that create β-sheet augmentation38. Most HECT ligases share the same composition of the catalytic loop with the catalytic cysteine preceded by a histidine and threonine and followed by a phenylalanine residue, which is functionally important (Supplementary Fig. 8a)11–13,42. In HECTD3, the loop composition is not conserved, and the catalytic cysteine is preceded by a serine and threonine residue (STCS instead of HTCF) (Supplementary Fig. 8a). Mutation of the catalytic loop residues to match those of other HECTs significantly reduced ubiquitin loading and ablated auto-ubiquitination activity (Supplementary Fig. 8b). We conclude that the catalytic loop is fine-tuned to the adjacent residues of each HECT ligase to optimise function.

For NEDD4 family members, the last four C-terminal amino acids have been shown to be functionally essential, harbouring a phenylalanine residue that is required for ubiquitin transfer as well as acidic residues that are proposed to serve as the catalytic base for target lysine deprotonation11–13,43,44. While the HECTD3 C-terminus has no sequence similarity to NEDD4’s, it harbours two acidic residues (E860 and E861) that could potentially perform the same function (Supplementary Fig. 8a). Deletion of the last four amino acids (HECTD3Δ4) did not abolish auto-ubiquitination activity, despite a slight reduction in ubiquitin loading (Supplementary Fig. 8c). We conclude that these residues do not function as HECTD3 catalytic bases but might still be important for ubiquitin orientation. Among other acidic residues near the active site, we identified D539 of the N-lobe and D855 of the C-terminal tail as well-positioned to fulfil catalytic functions (Fig. 4d). Mutations of either residue significantly reduced auto-ubiquitination while still maintaining trans-thiolation activity, albeit to a lesser extent (Fig. 4e, f). Additionally, increasing the pH of the reaction compensates the catalytic requirement for a base to increase lysine nucleophilicity and restores some auto-ubiquitination activity for both mutations (Supplementary Fig. 8d). Thus, both residues likely contribute to catalysis, with optimal target lysine positioning and deprotonation depending on the interplay of different acidic side chains in the active site.

HECTD3 mediates multi-monoubiquitination and formation of short polyubiquitin chains
HECTD3 is reported to form several different ubiquitin linkages in cells. To understand the molecular basis for chain linkage specificity, we analysed which type of ubiquitin chains are synthesised by HECTD3 in vitro. To probe linkage type we used ubiquitin mutants, where individual lysine residues, or all except one lysine are mutated to arginine. Additionally, we analysed activity with a lysine-less ubiquitin (K0) to test if the higher molecular weight bands observed during autoubiquitination are polyubiquitin chains or attachment of multiple mono-ubiquitin molecules. Interestingly, the HECT domain itself shows little preference for any linkage type (Fig. 5a and Supplementary Fig. 9a, b). This is surprising, as for some HECT ligases, the type of ubiquitin chains they synthesise is believed to be encoded in the C-lobe of the HECT domain45,46. In contrast, the full-length protein shows significant reduction of activity with the K11R mutant and to a lesser extent with K48R, with restored activity with the K11 only mutant (Fig. 5b and Supplementary Fig. 9b). Furthermore, ubiquitination assays with the K0 mutant showed reduced activity, especially with the full-length protein, suggesting that the higher molecular weight bands observed are polyubiquitin chains (Supplementary Fig. 9c). Finally, we investigated whether HECTD3 may form branched chains using Ub-clipping47, which analyse ubiquitin chain architecture to detect chain branching via an engineered viral protease and intact mass-spectroscopy. Both the HECT domain and the full-length protein show some branching activity but preferentially form linear chains (Fig. 5c). Taken together, our biochemical experiments with ubiquitin mutants suggest that HECTD3 forms K11-linked polyubiquitin chains with some K48 content. However, our HECTD3-Ub structure shows that ubiquitin K11 makes contacts with HECTD3 N848 (Fig. 4a), raising the possibility that its mutation may affect the overall activity observed in ubiquitination assays. To test this possibility, we carried out a E2-E3 transthiolation assay with the K11R mutant to analyse if the mutation may affect ubiquitin loading onto the E3. This experiment showed that HECTD3~UbK11R thioester intermediate formation is significantly reduced with the mutant, implying that the observed reduction of HECTD3 autoubiquitination with UbK11R does not indicate K11-linked chain specificity but rather the importance of K11 for efficient ubiquitin loading onto HECTD3 (Supplementary Fig. 9d). Furthermore, based on this observation, the K0 ubiquitin variant cannot be used to probe for polyubiquitin chain formation by HECTD3. Therefore, given the relatively modest shift in molecular weight observed in Coomassie-stained autoubiquitination assays, we suggest that HECTD3 mainly adds multi-mono ubiquitin onto itself together with some short polyubiquitin chains possibly containing K48 linkages.

PARP1 ubiquitination by HECTD3
Next we assessed substrate recognition and ubiquitination by HECTD3 to investigate if a given substrate can influence the type of chain it is modified with. First we aimed to validate substrates reported in the literature in vitro with purified proteins. While we could not detect any ubiquitination of Caspase-831 or Caspase-930, nor STAT334 by Coomassie staining, we detected a low level of STAT3 ubiquitination by western blot (Supplementary Fig. 10a–d). In contrast, PARP132, another proposed substrate, was robustly modified by HECTD3 (Fig. 6a). Autoubiquitination of HECTD3 and formation of di- and tri-Ub chains were detected in the Caspase-8, −9 and STAT3 lanes, but not with PARP1 indicating that substrate recognition suppresses auto-ubiquitination (Supplementary Fig. 10c, d). PARP1 plays a key role in the DNA damage response, during which its PARylation activity recruits DNA repair proteins to the site of damage48. HECTD3 was recently shown to promote the tumourigenesis of glioblastoma by modifying PARP1 with K63-linked chains to activate EGFR signalling32. We show that HECTD3 dependent modification of PARP1 required the presence of the region N-terminal to the core HECT domain and is only observed robustly in the context of the full-length protein, although we could observe small levels of mono-ubiquitination of PARP1 by the isolated HECT domain (Fig. 6a). As our previous experiments showed that ubiquitin mutants are not suitable to assess the chain linkage specificity of HECTD3, we instead used chain specific antibodies to assess PARP1 modification. These experiments show that HECTD3 as well as PARP1 are modified by K48-linked chains, but hardly any K63-linked chains as previously reported32 (Supplementary Fig. 11a). The E2 preference for UBE2D is maintained for PARP1 ubiquitination (Supplementary Fig. 11b), and Ub-clipping did not detect branched ubiquitin chains on PARP1 (Supplementary Fig. 11c). Finally, we determined which lysine residues on PARP1 are modified by HECTD3. Using mass-spectrometry, we identified six ubiquitination sites (K119, K346, K400, K409, K508 and K564) that all reside in the first half of the protein within the DBD, BRCT and WGR domains (Fig. 6b).

Our structural understanding of substrate recognition and presentation to the active site is currently limited due to the transient nature of E3-substrate complexes. Hence, to gain information about binding interfaces between PARP1 and HECTD3, we used cross-linking mass-spectrometry (XL-MS). We observed cross-links between HECT3’s DOC domain and the DBD of PARP1, confirming interaction domains identified in co-immunoprecipitation experiments32 (Fig. 6c). Additionally, we observe several cross-links between the HECT domain and the N-terminal portion of PARP1, with the interface spanning the DBD, BRCT and WGR domains. This agrees with the location of ubiquitination sites, that reside within that region and may explain why we observe some residual ubiquitination of PARP1 in the absence of the DOC: The HECT domain could provide a secondary binding site that stabilises the substrate bound complex and aid the optimal positioning for substrate ubiquitination. To support the XL-MS data, we employed Alphafold3 to predict the PARP1-HECTD3 complex structure. However, PAE (predicted alignment error) blots indicate a very low confidence in the interface prediction that also does not match the cross-link data (Fig. 6c) and thus did not provide additional insight into complex formation.

Although the DOC domain is found in other E3 ligases, how it recognises substrates is still poor understood. Most insights around substrate recognition stem from the DOC domain of the APC/C that recognises degron motifs on substrates49. To gain insight into substrate recognition, we analysed the evolutionary conservation of HECTD3 DOC domain (Fig. 6d, Supplementary Fig. 12a). We identified two potential residues that might be involved in substrate binding: Y252 and F256 are both surface exposed but not conserved suggesting that they are well placed to recognise specific substrates (Fig. 6d). Indeed, mutation of either amino acid resulted in significant reduction of PARP1 ubiquitination indicating their likely involvement in substrate recognition (Fig. 6e and Supplementary Fig. 12b). In contrast, auto-ubiquitination and HECTD3~Ub thioester formation were not affected by the mutations, indicating that the observed effect is due to decreased substrate recognition and not destabilisation of the E3 (Supplementary Fig. 12c, d).

In conclusion, we validated PARP1 as a bona fide substrate of HECTD3 which becomes modified with K48-linked ubiquitin chains on multiple lysine residues within the DBD, BRCT and WGR domains. Combining XL-MS with mutational analysis enabled us to determine binding sites on both proteins and identify two key residues on the DOC domain that contribute to substrate recognition.

Methods
Cloning, protein expression and purification
The codon optimised sequence of full-length human HECTD3 (1–861) was synthesised as gblocks (IDT) and ligated via Gibson assembly into pACEBAC1 vector with an N-terminal His6-fusion and a HRV3C protease cleavage site. All HECTD3 HECT constructs were further sub-cloned into pET49b vectors with an HRC3C protease cleavable GST-His6-tag via Gibson Assembly. The DOC domain (238–376) was cloned in the same manner. Mutations were introduced using QuickChange technique. pCMV5 FLAG PARP1 was obtained through the MRC PPU Reagents and Services facility (MRC PPU, College of Life Sciences, University of Dundee, Scotland) and used to sub-clone full-length PARP1 into pACEBAC1 vector with an N-terminal His6-fusion and a HRV3C protease cleavage site. Lbpro protease in the pET11d vector with His8-tag was kindly gifted by Kirby Swatek.
Expression and purification of E1 (UBA1) and E2 enzymes (UBE2D1/2/3 and UBE2L3) were performed as described previously61. Atto-labelling of ubiquitin and generation of UBED2A~UbAtto was done as described in ref. 61. Ubiquitin from bovine erythrocytes (Sigma, #U6253), used in all ubiquitination assays, was solubilized in 20 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP and purified by size-exclusion in the same buffer.
All HECTD3 HECT variants were expressed in ArcticExpress cells (DE3) competent cells (Agilent, #230192) according to manufacturer’s instructions. Briefly, cells were grown in LB media at 30°C until OD of 0.7 and expression was induced with 500 μM IPTG and performed for 24 h at 13°C. Cells were harvested and resuspended in IMAC A buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole) supplemented with 10 mM MgCl2, 5% glycerol (v/v), DNAse (1 μg/ml) and protease inhibitor cocktail (Roche). Lysis was done by sonication and lysate was cleared by centrifugation (1 h at 50,000 × g, 4°C). Lysate was subsequently loaded onto HisPur™ Ni-NTA Resin in IMAC A buffer and after elution with 250 mM imidazole, supplemented with homemade HRC 3C protease. Reverse affinity purification was done to remove tags and the protein further purified via SEC (HiLoad Superdex 16/600 75 pg; Cytiva) into 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol. The DOC domain was purified in the same manner.
Full-length HECTD3 was expressed in Sf9 cells using the baculovirus transfer vector pACEBac1 system. Cells were cultured at 28°C in SF900 III serum-free media (Invitrogen). Expression was performed by infecting 2×106 cells/ml with 1 ml of high-titer baculovirus for 72 h at 28°C. Cells were harvested and lysed on ice for 1 h in lysis buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 20 mM imidazole, 5% glycerol, 0.5% Triton X-100 (v/v), 10 mM MgCl2, 0.1 mM AEBSF, DNAse (1 μg/ml) and protease inhibitor cocktail (Roche)). Lysate was cleared by centrifugation and further purified via NiNTA chromatography similarly to HECT constructs, with the final purification step being SEC (HiLoad Superdex 10/300 200 pg; Cytiva) into 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol. Lbpro protease was expressed and purified as described in ref. 62.
PARP1 was expressed in Sf9 cells similar to the expression of HECTD3 and purified as previously described63 with the final purification step being SEC (HiLoad Superdex 10/300 200 pg; Cytiva) into 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol.
Samples for SAXS and cryo-EM were expressed and purified similar as described above with modifications. After NiNTA elution, 3C-protease was added to the eluted protein and dialysed overnight against 25 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mM TCEP and 2.5% glycerol. The cleaved protein was then loaded onto a HiTrap FF Q anion exchange column equilibrated with 25 mM HEPES pH 7.5, 100 mM NaCl, 0.5 mM TCEP and 2.5% glycerol and eluted with a 100–500 mM NaCl gradient. Peaks corresponding to HECTD3 were concentrated to ~7 mg/ml and loaded onto a Superdex S200 Increase 3.2/300 equilibrated with 50 mM Na-phosphate pH 7, 200 mM NaCl, 0.5 mM TCEP and 2% glycerol.
For preparation of Ub-loaded HECTD3, 0.5 mg of HECTD3 from the ion-exchange step and 1.5 mg of Ubiquitin-propargylamide were mixed for 2 h at 20°C. The reaction was then concentrated to ~100 µl and loaded onto a Superdex S200 Increase 3.2/300 equilibrated with 50 mM Na-phosphate pH 7, 200 mM NaCl, 0.5 mM TCEP and 1.5% glycerol.

Ubiquitination assays
Auto-ubiquitination assays were performed in 25 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2 in the presence of 500 nM E1, 2 μM of respective E2s, 50 μM ubiquitin, 10 mM ATP and 4 μM E3. Reactions were performed at 30°C at 750 rpm shaking and samples were taken at time-points indicated in the figure panel, supplemented with 2x reducing LDS sample buffer and analysed on SDS-PAGE.
For substrate ubiquitination assays, conditions were slightly modified, and assays were performed using 100 nM E1, 2 μM of UBE2D3, 50 μM ubiquitin, 10 mM ATP, 1 μM E3 and 3 μM substrates. Other reactions conditions remained the same and samples were taken as described above. Reactions were either analysed using SDS-PAGE directly, or via western blot, using the protocol described for the E2 scan analysis. Western blot analysis was done against ubiquitin using the mouse antibody Ubi-1 (Invitrogen, 13–600, 1:1000) and FK2 (Sigma, ST1200, 1:1000).

E3 trans-thiolation assay
For general E3 trans-thiolation activity assays with ubiquitin, UBE2DA~UbAtto was generated as described in ref. 61. The assay was performed in 25 mM HEPES pH 7.5, 150 mM NaCl by mixing 1 μM of UBE2D1~UbAtto either with 5 μM E3 in assays comparing FL and HECT ligase or 10 μM E3 in assays comparing different HECT mutants. Samples were incubated at 30°C and samples were taken after time-points indicated in the figure panel by mixing with 2x LDS sample buffer (non-reducing) and flash freezing in liquid N2. Samples were then analysed with SDS-PAGE and imaged in the Chemdoc imager (Bio-Rad) for in gel fluorescence. For E3 trans-thiolation assays with ubiquitin or the K11R mutant, UBE2D3~UbAtto was generated in situ by mixing 0.5 μM E1, 10 μM of UBE2D3, 50 μM UbATTO (WT or K11R) and 10 mM ATP in 50 mM HEPES pH 7.5, 150 mM NaCl and 10 mM MgCl2. The reaction was incubated a 30°C for 15 min then quenched with EDTA. The trans-thiolation assay was performed by mixing the solution of UBE2D3~UbATTO diluted 10x with 5 μM of HECTD3 in 50 mM HEPES pH 7.5, 150 mM NaCl and incubated at 30°C. Samples were analysed as described above.

Generation of ubiquitin-propargylamide (Ub-PA)
Ub-PA was prepared using an intein-tagged ubiquitin (1-75) construct. The protein was expressed in BL21 cells grown in LB media until OD of ~0.8 was reached and expression was induced with 500 μM IPTG overnight at 20°C. Cells were harvested by centrifugation and pelleted cells were resuspended in wash buffer (25 mM MES pH 6.5, 100 mM NaCl) supplemented with 10 mM MgCl2, DNAse and protease inhibitor. Cells were lysed and centrifuged similarly to HECT constructs and lysate was loaded on chitin resin (New England BioLabs) and allowed to bind at room-temperature for 2–3 h. Resin was washed extensively with wash buffer and eluted in the presence of 150 mM MESNA overnight at room-temperature to generate Ub-MESNA. Ub-MESNA was further purified by SEC using HiLoad Superdex 16/600 75 pg (Cytiva) in wash buffer. To generate Ub-PA, the pH was adjusted to 8.5 using HCl and propargylamine was added at approximately 1000x excess. Reaction was performed at RT for 4–6 h and afterwards dialysed against 50 mM HEPES pH 8, 150 mM NaCl. Generation of Ub-PA was confirmed by in house intact mass-spectrometry and labelling efficiency ranged from 60 to 70%.

Ub-PA reactivity assays
3 μM of either HECTD3 HECT or full-length HECTD3 was incubated with 100 μM Ub-PA in 25 mM HEPES pH 7.5, 150 mM NaCl at 30°C for indicated time-points. Reactions were quenched by the addition of 2x SDS loading buffer and analysed using SDS-PAGE.

SEC-MALLS
Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) was performed with 120 μl 0.45 μm-filtered protein samples at concentrations of 0.5, 1, 2 and 4 mg/ml of the HECT and full-length HECTD3 using a Superdex 200 Increase 10/300 column on a Jasco HPLC system in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP and 3 mM NaN3. All runs were performed with a flow-rate of 1 ml/min and recorded using a DAWN-HELIOS-II laser photometer (Wyatt Technology), and an OPTILAB differential refractometer (Wyatt Technology). Data were analysed using the Astra software package.

E2 scan
The E2 scan was performed using the commercially available E2 scanTM Kit version 2 (UBIQUENT) according to the manufacturer’s instructions with 1 μM E3 at 30°C for 1 h. Following SDS-PAGE, ubiquitination was detected via western blot. Blotting was done using the Trans Blot Turbo system (Bio-Rad) and membranes were blocked for 1 h at room-temperature in 5% milk in PBS with 0.1% Tween-20, before incubation with anti-Ub antibody (Invitrogen 13–600, 1:1000 in blocking solution), followed by washing steps and incubation with HRP-coupled anti-mouse antibody (Cell Signalling Technology #7076, 1:2000 in blocking solution). Membranes were imaged using commercial ECL solution (Amersham) on the ChemiDoc imager (Bio-Rad).

Detection of ubiquitin chains using selective antibodies
To analyse the chains formed by HECTD3, samples of auto-ubiquitination or substrate-ubiquitination assays (performed as described above) were analysed on SDS-PAGE, followed by western blot with different antibodies. Blotting was done using the Trans Blot Turbo system (Bio-Rad) and membranes were blocked for 1 h at room-temperature in 5% milk in PBS with 0.1% Tween-20, before incubation with either anti-Ub antibody (Ubi-1; Invitrogen 13–600, 1:1000 in blocking solution), K48 specific antibody (Abcam ab140601, 1:1000 in blocking solution) or K63 specific antibody (Abcam ab179434, 1:1000 in blocking solution) over-night at 4°C. Washing steps with PBS-T were followed by incubation with HRP-coupled anti-mouse antibody (Cell Signalling Technology #7076, 1:2000 in blocking solution) or anti-rabbit antibody (Cell Signalling Technology #7074, 1:2000 in blocking solution). Membranes were imaged using commercial ECL solution (Amersham) on the ChemiDoc imager (Bio-Rad).

Ub-clipping
Samples for Ub-clipping were generated using conditions described for auto- or substrate-ubiquitination assays with the slight modification of a 2 h incubation step to ensure maximum ubiquitin usage. Reactions were quenched by the addition of 20 mM EDTA for 10 min. Lbpro protease was added to a final concentration of 5 μM and samples were incubated at 30°C for 4 h. Samples were then flash frozen and analysed with intact-mass spectrometry.
For intact-mass spectrometry, denatured proteins were injected onto a C4 BEH 1.7 μm, 1.0 × 100 mm UPLC column using an Acquity I class LC (Waters, UK). Proteins were eluted with a 15 min gradient of acetonitrile (2% v/v to 80% v/v) in 0.1% v/v formic acid using a flow rate of 50 μl/min. The analytical column outlet was directly interfaced via an electrospray ionisation source, with a time-of-flight (TOF) mass spectrometer (BioAccord, Waters, UK). Data was acquired over a m/z range of 300–8000, in positive ion mode with a cone voltage of 40 V. Scans were summed together manually and deconvoluted using MaxEnt1 (Masslynx, Waters, UK).

Cryo-EM
Sample preparation and data collection. Cryo-EM grids were prepared using freshly purified protein samples. Fractions from the peak of size exclusion chromatography were diluted to 0.92 mg/ml for HECTD3 and 1.5 mg/ml for HECTD3-Ub in their respective gel filtration buffers. Prior to grid preparation, octyl glucoside was added to a final concentration of 0.1%. Four microliter of each sample was applied to non-glow discharged C-flat holey carbon grids (400 mesh, 1.2/1.3 μm, Au) at 4°C and 100% humidity. Samples were immediately blotted for 5 s and vitrified by plunging into liquid ethane using a Vitrobot Mark IV (FEI-Thermo Fisher).
Cryo-EM data were collected on an in-house 300 keV Titan Krios transmission electron microscope (FEI-Thermo Fisher) equipped with a Falcon IV detector and a Selectris energy filter at a calibrated pixel size of 0.95 Å/pix. For both samples, 31 movies were recorded in electron event representation (EER) format at total electron doses of 39.76 for HECTD3 and 40.99 e-/Å2 for HECTD3-Ub. The defocus ranged from −3.0 to −1.2 μm for HECTD3 and −3.0 to −0.6 μm for HECTD3-Ub data acquisition. Automated data collection was performed with EPU (Thermo Fisher), resulting in the recording of 25,560 movies for HECTD3 and 37,160 movies for HECTD3-Ub.
Cryo-EM data processing. Detailed workflows for each dataset are illustrated in Supplementary Figs. 5 and 7. Initial data processing steps were similar for HECTD3 and HECTD3-Ub. Movies were drift-corrected and summed with Relion implementation of MotionCor264. Contrast transfer function (CTF) estimation was performed with Gctf using 5×5 patches. Micrographs were discarded if they had an estimated resolution lower than 6 Å, negative figure of merit, or astigmatism higher than 1000 Å for HECTD3 and 1500 Å for HECTD3-Ub. Image processing was performed using Relion 4.065 and cryoSPARC66, with UCSF Pyem67 used to convert particle metadata between software packages.
For HECTD3, initial particle picking involved manually selecting 1000 particles from 50 micrographs to generate a training model in Cryolo68, followed by particle picking for the entire dataset. The initial picked particles (1,295,970) were extracted at 3.74 Å/px (4-fold downsampling) and subjected to interactive 2D classification to eliminate low-quality particles. The remaining particles (898,749) were used for ab initio reconstruction across 5 classes followed by non-uniform refinement. The most promising class (275,111) underwent an additional round of 2D classification in Relion with selected particles (202,468) subsequently employed in 3D classification with 4 classes with image alignment (T=4, 180 Å mask) and the initial reference map low-pass filtered to 60 Å. Particles from the largest class (74,784) were re-extracted with no downsampling and subjected to a round of ab initio reconstruction using 2 classes followed by non-uniform refinement. The predominant class particles (45,474) were extracted (3.8 Å/px, 4-fold downsampling) and used for neural network-based particle picking in Topaz69. The identified particles (3,537,942) were extracted (3.8 Å/px, 4-fold downsampling) and underwent junk particles removal by 2D classification to yield 874,598 particles. To separate HECTD3 conformations, after 2D classification the particles were extracted with 2 distinct box sizes (no downsampling and 1.90 Å/px 2-fold downsampling) and subjected to multiple rounds of ab initio reconstruction and heterogeneous refinement with each iteration employed to select the most promising classes. This approach successfully identified particles representing both conformational states, which were subsequently consolidated, de-duplicated, and processed independently. The final particle ensemble for the “inverted-T” conformation (75,003) underwent Bayesian polishing and non-uniform refinement, resulting in a map at 6.38 Å resolution. Conversely, the L-conformation particles (105,628) were Bayesian polished and processed through a final ab initio reconstruction with 3 classes, followed by heterogeneous refinement. Two representative classes were selected for a final non-uniform refinement, generating a map at 6.06 Å resolution.
For HECTD3-Ub, Cryolo picked particles (3,638,210) were extracted at 3.8 Å/px (4-fold downsampling) and cleaned through multiple 2D classification iterations. The remaining high-quality particles were used to train Topaz neural network for subsequent particle picking. These particles underwent cleaning via 2D classification, and two rounds of ab initio reconstruction followed by heterogenous refinement across 3 classes. The most promising particle subsets were independently subjected to 3D classification in Relion, employing 3 and 7 classes, respectively. The best classes from each 3D classification were merged (741,885), extracted without downsampling, and imported in cryoSPARC for three consecutive rounds of ab initio reconstruction and heterogeneous refinement. The best particles were imported in Relion, re-extracted with a larger box size (296 px) without downsampling and underwent Bayesian polishing and CTF refinement. Finally, they were reimported in CryoSPARC to be subjected to local and global CTF refinement and a final round of ab initio reconstruction and heterogeneous refinement. To explore the available HECTD3-Ub conformational space, the best particle ensemble (342,589) was subjected to 3D variability analysis40 with a 5 Å resolution filter and 3 orthogonal principal modes, followed by a non-uniform refinement step to yield a final map of 3.04 Å resolution.
Model building and refinement. Local resolution estimation for both HECTD3 and HECTD3-Ub reconstructions was performed in cryoSPARC using FSC thresholds of 0.143 and 0.5. Maps filtered to FSC=0.5 based on local resolution estimates were used for model building and refinement.
For the HECTD3-Ub structure, initial coordinates were generated by rigid-body docking of the HECTD3 AlphaFold model (excluding the DOC domain), ubiquitin (PDB 1UBQ), and our DOC domain crystal structure (residues 237-374) into the locally-filtered cryo-EM map using UCSF ChimeraX70. Iterative cycles of manual model building in Coot71 and real-space refinement in Phenix72 were performed until satisfactory stereochemistry and model-to-map fit were achieved, as assessed by Phenix validation tools and MolProbity73. EMReady-sharpened maps74 were utilised to facilitate chain tracing and model building. The DOC domain was treated as a rigid body during refinement.
For the apo HECTD3 model building, initial coordinates were obtain by separately docking the HECTD3 (lacking DOC and C-lobe regions), the C-lobe (residues 748-849) from the refined HECTD3-Ub, and the DOC domain crystal structure (residues 237-374) into their respective cryo-EM density regions of the L-shaped and “inverted-T” HECTD3 conformations using ChimeraX70. The DOC domain and C-lobe were maintained as rigid bodies, while the remaining HECTD3 underwent iterative manual model adjustment in Coot followed by real-space refinement in Phenix. Secondary structure restraints were applied during Phenix refinement to preserve proper geometry, with final model validation performed by MolProbity. All structure representations and movies were generated using UCSF ChimeraX. Model refinement statistics are provided in Supplementary Table 1. Coordinates and maps are deposited for HECTD3 in the L-conformation with the codes 9R8T and EMD-53836, for HECTD3 in the inverted T-conformation with the codes 9R94 and EMD-53852, and for HECTD3-Ub with the codes 9R85 and EMD-53804.

X-ray crystallography
Sitting-drop method was used to crystallise HECTD3 DOC domain (residues 238-377) in 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP at a concentration of 13.5 mg/ml. Commercially available crystallisation screens were dispensed at 20°C using an automated NT8 crystallisation robot (Formulatrix). Crystals grew from a 100 nL protein solution plus 100 nL reservoir made of 0.1 M HEPES pH 7.5, 30% (w/v) PEG 400, 5% PEG 3000 and 10% glycerol. For data acquisition, crystals were cryoprotected with the mother liquor alone. Diffraction data were collected on beamline MASSIF-3 at the ESRF (Grenoble), processed using XDS75 and merged and scaled using AIMLESS76. The structure of the domain was solved by molecular replacement using the AlphaFold structure of the HECTD3 DOC domain as templates in Phenix Phaser. Models were iteratively improved by manual building in Coot and refined using Phenix. Coordinates and structure factors are deposited in the Protein Data Bank under accession code 9R6V. Further details on data collection and refinement statistics are summarised in Supplementary Table 2.

SAXS
SEC-SAXS data were collected at the B21 beamline at Diamond Light Source (DLS, UK). Samples of HECTD3 at 8.9 mg/ml and HECTD3-Ub at 5.5 mg/ml were injected onto a Superdex 200 3.2×300 column and eluted at a flow rate of 0.075 ml/min at 15°C with 3 s exposures. Frames were continuously collected during the fractionation process, with those captured before the void volume averaged and subtracted from the elution profile signal to account for background scattering. Data reduction, subtraction, and averaging of frames within the SEC peak, characterised by a constant Rg, were carried out using the software ScÅtter IV (www.bioisis.net). Scattering curves, analyzed using the ATSAS package77, are reported as a function of the angular momentum transfer q=4π/λ sin θ, where 2θ represents the scattering angle and λ the wavelength of the incident beam. The statistical results are summarised in Supplementary Table 3.

Crosslinking (XL) MS
15 μM complex (HECTD3 and PARP1) was incubated with DSBU (disuccinimidyl dibutyric urea) at 100 molar excess for 2 h on ice. The reaction was quenched with 20 mM TRIS pH 7.5. The crosslinked complex was reduced with 10 mM dithiothreitol and alkylated with 50 mM iodoacetamide. It was then digested with trypsin at an enzyme-to-substrate ratio of 1:100, for 1 h at RT and further digested overnight at 37°C following addition of trypsin at a ratio of 1:20. The peptide digests were then fractionated batch-wise by high pH reverse phase chromatography on microspin TARGA C18 columns (Nest Group Inc, USA), into six fractions (10 mM NH4HCO3/10% (v/v) acetonitrile pH 8.0, 10 mM NH4HCO3/15% (v/v) acetonitrile pH 8.0, 10 mM NH4HCO3/20% (v/v) acetonitrile pH 8.0, 10 mM NH4HCO3/30% (v/v) acetonitrile pH 8.0, 10 mM NH4HCO3/40% (v/v) acetonitrile pH 8.0 and 10 mM NH4HCO3/80% (v/v) acetonitrile pH 8.0). The fractions (120 μL) were evaporated to dryness in a CentriVap concentrator (Labconco, USA) prior to analysis by LC-MS/MS.
Lyophilised peptides were resuspended in 1% (v/v) formic acid and 2% (v/v) acetonitrile and analysed by nano-scale capillary LC-MS/MS using a Vanquish Neo UPLC (Thermo Scientific Dionex, USA) to deliver a flow of approximately 250 nL/min. A PepMap Neo C18 5 μm, 300 μm × 5 mm nanoViper (Thermo Scientific Dionex, USA) trapped the peptides before separation on a 50 cm EASY-Spray column (50 cm × 75 μm ID, PepMap C18, 2 μm particles, 100 Å pore size: Thermo Scientific, USA). Peptides were eluted with a gradient of acetonitrile over 90 min. The analytical column outlet was directly interfaced via a nano-flow electrospray ionisation source, with a quadrupole Orbitrap mass spectrometer (Orbitrap Exploris 480, Thermo Scientific, USA). MS data were acquired in data-dependent mode using a top 10 method, where ions with a precursor charge state of 1+ and 2+ were excluded. High-resolution full scans (R=60,000, m/z 380–1800) were recorded in the Orbitrap followed by higher energy collision dissociation (HCD) (stepped collision energy 30 and 32% Normalised Collision Energy) of the 10 most intense MS peaks. The fragment ion spectra were acquired at a resolution of 15,000 and a dynamic exclusion window of 20 s was applied.
For data analysis, Xcalibur raw files were converted into the MGF format using Proteome Discoverer version 2.3 (Thermo Scientific) and used directly as input files for MeroX78. Searches were performed against an ad hoc protein database containing the sequences of the proteins in the complex and a set of randomised decoy sequences generated by the software. The following parameters were set for the searches: maximum number of missed cleavages: 3; targeted residues K, S, Y and T; minimum peptide length 5 amino acids; variable modifications: carbamidomethylation of cysteine (mass shift 57.02146 Da), methionine oxidation (mass shift 15.99491 Da); DSBU modified fragments: 85.05276 Da and 111.03203 Da (precision: 5 ppm MS and 10 ppm MS/MS); False Discovery Rate cut-off: 5%. Resulting Cross-links were visualised in xiView79.

Identification of ubiquitination sites on PARP1
Reconstituted proteins were resolved by SDS-PAGE, stained with Coomassie G-250 and the resultant protein gel bands corresponding to ubiquitinated species of PARP1 excised and analysed by lc-ms/ms. Briefly, the excised protein gel pieces were placed in individual 1.5 ml low bind Eppendorf tubes and destained with 50% v/v acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated with 55 mM iodoacetamide. After alkylation, proteins in gel pieces were digested with 6 ng/mL trypsin or Asp-N overnight at 37°C. The resulting peptides were extracted in 1% v/v formic acid, 2% v/v acetonitrile and the combined supernatants dried in a vacuum centrifuge before resuspending in 0.1% trifluoroacetic acid (TFA). The digests (1 to 10 μl) acidified to a final concentration of 0.1% TFA, were analysed by nano-scale capillary lc-ms/ms using an UltiMate 3000 nanoRSLC HPLC (Thermo Scientific, San Jose, USA) to deliver a flow of approximately 7 μl/min of 0.1% TFA onto a 75 μm × 2.0 cm mm Acclaim PepMap 100 C18 trap column (Thermo Fisher Scientific) before the trap being switched to elute at 0.25 μl/min through a 50 cm × 75 μm EASY-Spray C18 column. A 90′ gradient of 9 to 25% B over 37′ and then 25 to 40% B over 18′ was used followed by a short gradient to 100% B and back down to 9% B and a 20′ equilibration in 9% B [A=2% acetonitrile (ACN), 0.1% formic acid (FA); B=80% ACN, 0.1% FA].
The analytical column outlet was directly interfaced with a hybrid dual pressure linear ion trap mass spectrometer (Orbitrap Lumos, Thermo Scientific, San Jose, USA). Data dependent acquisition was carried out, using a resolution of 120,000 for the full MS survey scan, followed by MS/MS in “Top S” mode in the linear ion trap. MS spectra were collected over a m/z range of 300–1500. MS/MS scans were collected with the standard AGC target, dynamic maximum injection time mode, isolation window at 1.2 m/z and 32% normalised HCD collision energy. All raw files were processed with MaxQuant 2.4.2.0 using standard settings and searched against a UniProt Human Reviewed KB database with the Andromeda search engine integrated into the MaxQuant software suite80,81. Enzyme search specificity was Trypsin/P or AspN for the endoproteinases. Up to two missed cleavages for each peptide were allowed. Carbamidomethylation of cysteines was set as a fixed modification with oxidised methionine, protein N-acetylation and GlyGly additions to lysine residues considered as variable modifications. The search was performed with an initial mass tolerance of 6 ppm for the precursor ion and 0.5 Da for MS/MS spectra. The false discovery rate was fixed at 1% at the peptide and protein level. Statistical analysis was carried out using the Perseus module (v1.4.0.2) of MaxQuant.

AlphaFold prediction
Protein structure prediction was performed using The Francis Crick Institute local installation of AlphaFold382. The software was run with default parameters on the Francis Crick Institute high-performance computing cluster. Input sequences were provided in FASTA format, and the predictions were generated using the full database mode. PAE blots were rendered in ChimeraX.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Huber et al. presents a comprehensive biochemical and structural analysis of HECTD3, a member of the HECT E3 ligase family. While HECT ligases are central regulators of ubiquitin-mediated signaling, their mechanisms of substrate selection and chain specificity remain incompletely understood. HECTD3 has been implicated in inflammation, apoptosis, infection, and cancer progression, yet its structure and enzymatic activity have remained largely uncharacterized. The authors combine biochemical ubiquitination assays, E2 profiling, linkage type analysis, cryo-EM and crystallography, SAXS, XL-MS, and mutational studies to provide the first full-length structural snapshots of HECTD3 in both apo and ubiquitin-loaded states. They reveal a novel N-terminal fold, identify unusual E2 preferences (favoring UBE2D family members over UBE2L3), and present evidence suggesting that HECTD3 catalyzes mainly K11- and K48-linked ubiquitin chains. The study further validates PARP1 as a substrate, maps ubiquitination sites, and proposes mechanistic roles for specific residues in chain-type determination and substrate binding.\n\nThe manuscript delivers a rigorous and technically impressive characterization of HECTD3, combining structural biology, biochemistry, and substrate validation to provide new insights into this poorly understood ligase. The cryo-EM reconstructions of full-length HECTD3, supported by crystallography of the DOC domain and SAXS data, are of particular value, revealing two unique N-terminal folds (D3NA and D3NB) and conformational flexibility between L- and inverted T-states characteristic of HECT ligases. The structural work is highly convincing and, aside from a minor figure presentation issue, excellent. The biochemical analyses, however, raise concerns that require further clarification and more cautious interpretation.\n\n1) PARP1 binding and ubiquitination\nThe XL-MS data supporting PARP1 interaction with HECTD3 are strong, but the biochemical validation is less convincing. In Figure 6a, the Coomassie-stained SDS-PAGE shows only a modest shift in PARP1 molecular weight, suggesting attachment of few ubiquitin molecules, whereas the western blot in Supplementary Figure S10c shows distinct higher-molecular-weight species. It would be helpful if the authors clarified whether PARP1 is mainly multi-mono-ubiquitinated or polyubiquitinated. Do the DOC domain mutants (Y252A, F256A) abolish this higher-molecular-weight species in western blotting, and do they also impair HECTD3 auto-ubiquitination? Furthermore, can the authors exclude that these mutations interfere with E2–E3 charging? Finally, is the unusual preference for UBE2D E2s over UBE2L3 also observed in PARP1 ubiquitination assays?\n\n2) Reported substrates not ubiquitinated in vitro\nThe failure to ubiquitinate caspase-8, caspase-9, or STAT3 in vitro is surprising, since these proteins are reported in vivo substrates. Could this reflect a requirement for post-translational modifications or adaptor proteins absent in the assay system? For example, phosphorylation of HECTD3 at Thr157 has been reported as essential for caspase-9 ubiquitination, and MALT1 could act as an adaptor for STAT3 or caspase-8. In addition, Supplementary Figure 10 shows that HECTD3 auto-ubiquitination is absent in the presence of caspase-8 and caspase-9, which is puzzling since both are apparently no suitable substrates competing with the former activity; an observation that requires further explanation. The authors should add a statement to the discussion addressing why the majority of reported substrates were not modified under their assay conditions. Ultimately, the idea that a constitutively active, and thus uncontrolled, E3 ligase is present in the cytosol is to some extent worrisome. Is it possible that an additional layer of regulation that restricts HECTD3 from futile or even detrimental ubiquitination events is present in vivo. Although a verification of such regulatory mechanisms is most likely beyond the scope of this study, the authors might still want to include some of their thoughts in the discussion.\n\n3) Ubiquitin chain type\nThe conclusion that HECTD3 primarily catalyzes K11- and K48-linked chains is not fully convincing. Band patterns in Figure 1b and 1c suggest attachment of only a few ubiquitin molecules rather than chain elongation. In UbiCRest assays, residual ubiquitin signals after Cezanne or OTUB1 treatment could reflect priming ubiquitins rather than minor alternative linkages as the authors suggest. Moreover, OTUB1 treatment generates a strong, apparently monoubiquitinated species that persists even with Cezanne co-treatment, possibly pointing to an experimental artifact rather than linkage specificity? Finally, UB-clipping shows that only ~10% of ubiquitin carries two di-glycine remnants, further supporting the idea that HECTD3 primarily mediates attachment of multiple mono-ubiquitination or short chains rather than that of defined long K11 or K48 polyubiquitin chains. Correct?\n\n4) Interpretation of structural data\nThe high-resolution structure of the HECTD3–Ub intermediate suggests that ubiquitin K11 contributes to the interface with the C-lobe. However, it is unclear how much of this interaction is perturbed by K0 or K11R mutants. Could assays characterizing ubiquitin chains be blurred by introduced mutations?\n\n5) Domain representation\nThe topological description of the D3NA and D3NB domains is difficult to follow and not well presented in Figure 4d. The claim that residues 45–156 form “eight beta-sheets” sounds like a massive domain and should be replaced by “eight b-strands”. Secondary structure assignments are not always convincing (e.g. β-strand 4), and not all strands are labeled in the figure. A clearer illustration with D3NA and D3NB colored differently, and consistent color coding with Supplemental Figure S1, would be better.", "reviewer_2": "Huber, Rittinger et al. present a comprehensive structure-function analysis of HECTD3 E3 ligase. HECTD3 is one of the 28 human E3 ligases that all contain a C-terminal catalytic HECT domain, but differ in their N-terminal substrate binding and regulatory domains. In cells, HECTD3 was shown to ubiquitinate Caspase-9, PARP1, c-MYC and STAT3. The PDB holds over 200 structures of HECT E3s, as complexes, full-length proteins, or domains. But before now, there were no structures of HECTD3.\n\nMost of the work is a standard set of protein quality and E3 ligase test experiments. The researchers apply SEC-MALS to show HECTD3 is mainly monomeric. Use of the UBIQUENT E2scanTM kit showed most auto-ubiquitination with UBE2D2 and UBE2D3. The researchers do not find indication of auto-inhibition from testing reactivity with Ub-PR. There is not a single ubiquitin chain linkage formed in vitro. Low resolution cryo-EM structures of HECTD3 show the HECTD3 N-terminal domain and inverted T- and L-shaped HECT domain. Use of Ub-PR afforded model building for a HECTD3-Ub intermediate. At the end of the works, the researchers test ubiquitination of the proposed substrates. PARP1 was ubiquitinated in their hands. The researchers performed XL-MS and observed cross-links between PARP1 DBD and HECTD3 DOC domain. Cross-links were also observed between PARP1 NTDs and HECTD3 HECT domain.\n\nSummary: The study provides new knowledge of the E3 ligase HECTD3. The paper is very well-written. The figures in the main text are clear and very aesthetically-pleasing. The novelty comes from the focus on HECTD3. There are not many structures of HECT E3 complexes with substrates so there would be potential for novelty if the authors added a cryo-EM structure of the complex with PARP1. Otherwise, the work mainly extends to HECTD3 what is known from a large body of work on other HECT E3s. Below are specific points to be addressed in order of Figures.\n\nFigure 1. The HECTD3 E2 preference for UBE2D family members is not unique. Many papers in the last years have shown TRIP12 and UBR5 use UBE2D family members.\n\nFigure 2. It is unclear from the experiments if HECTD3 is being tested for a preferred activity. It is not clear what percentage of the products are monoubiquitin additions and what percentage are ubiquitin chains. It is also unclear if the auto-ubiquitination of the HECT domain alone is on the same site as FL HECTD3. It is also unclear if this HECT domain reaction proceeds in cis or in trans. Since the researchers cannot conclude if the K11R or K48R mutations have effects beyond linkage they should perform western blotting with linkage-specific antibodies if they wish to conclude preferences. Otherwise, the authors should qualify their conclusions about any linkage specificity for HECTD3.\n\nFigure 3. The researchers should consider removing “highly” from the label since the DOC domain is visible in the maps. It is not clear that data support the conclusion that the DOC domain is “highly” flexible compared to the other domains.\n\nFigure 5e and f. Since the mutations affect E3 loading, the researchers cannot rule out that over multiple cycles the impaired activity would not contribute a lot to the effects on auto-ubiquitination. The conclusions about mechanism cannot be drawn without performing Michaelis-Menten enzyme kinetics and pKa measurements. Those experiments would strengthen the conclusions, but they will not affect the novelty of the paper. So the authors could simply qualify their conclusions.\n\nFigure 6. It is difficult to compare PARP ubiquitination and HECTD3 auto-ubiquitination in this gel system. The bands are too compressed. It would be better to separate by a different gel system or analysis by western blotting.\n\nPanel e. The DOC domain Y252A and F256A mutants look like they have general problems. The band below the E2 is lost. There is not FL auto-ubiquitination even at 60 minutes. The authors need experiments showing that the effects are only on substrate binding. The authors should show the cryo-EM data for these residues as an indicator of model accuracy since the interaction with PARP1 is the most novel part of the work.\n\nSupplementary Figure S1. The statement HECTD3 is highly conserved across species should be reconsidered. The sequence alignment only shows vertebrate species.\n\nSupplementary Figure S2.\nThe researchers clearly demonstrate that HECTD3 is active with UBE2D family members. They should temper their conclusions however. Studying auto-ubiquitination at 60 minutes for the E2 scan could be misleading because there are a lot of steps other than transthiolation occurring by this point in the experiment.\n\nThe – and + labels in panel d are confusing. These look to refer to the E2 added but they probably refer to HECTD3. HECTD3 needs to be added to that line.\n\nSupplementary Figure S3. Is it difficult to see HECTD3 auto-ubiquitination in this gel system. The bands are compressed so it is difficult to differentiate effects of the lysine mutants.\n\nSupplementary Figure S10. The distances to the ubiquitination sites need to be shown if this is to support accuracy of the model.\n\nSupplementary Figure S11. This figure could be more clear. More depthcue or use of surface representation could be help."}, "author_response": "We thank the reviewers for their careful evaluation of our manuscript and their insightful comments and suggestions. To respond to the concerns raised we have carried out additional experiments and made significant changes to the manuscript. These changes include a re-evaluation of the chain linkage specificity detected, more detailed analysis of the DOC domain mutants, analysis of the autoubiquitination sites on HECTD3, investigation into cis or trans ubiquitination and a more detailed analysis of HECTD3 activity with UBE2D versus UBE2L3. To incorporate these changes in a logical manner we have slightly rearranged the sequence of reported results.\n\nWe like to thank the reviewers for their suggestions which we believe have significantly improved the manuscript. Please find below detailed answers to individual points raised.\n\nReviewer #1 (Remarks to the Author):\nThis manuscript by Huber et al. presents a comprehensive biochemical and structural analysis of HECTD3, a member of the HECT E3 ligase family. While HECT ligases are central regulators of ubiquitin-mediated signaling, their mechanisms of substrate selection and chain specificity remain incompletely understood. HECTD3 has been implicated in inflammation, apoptosis, infection, and cancer progression, yet its structure and enzymatic activity have remained largely uncharacterized. The authors combine biochemical ubiquitination assays, E2 profiling, linkage type analysis, cryo-EM and crystallography, SAXS, XL-MS, and mutational studies to provide the first full-length structural snapshots of HECTD3 in both apo and ubiquitin-loaded states. They reveal a novel N-terminal fold, identify unusual E2 preferences (favoring UBE2D family members over UBE2L3), and present evidence suggesting that HECTD3 catalyzes mainly K11- and K48-linked ubiquitin chains. The study further validates PARP1 as a substrate, maps ubiquitination sites, and proposes mechanistic roles for specific residues in chain-type determination and substrate binding.\n\nThe manuscript delivers a rigorous and technically impressive characterization of HECTD3, combining structural biology, biochemistry, and substrate validation to provide new insights into this poorly understood ligase. The cryo-EM reconstructions of full-length HECTD3, supported by crystallography of the DOC domain and SAXS data, are of particular value, revealing two unique N-terminal folds (D3NA and D3NB) and conformational flexibility between L- and inverted T-states characteristic of HECT ligases. The structural work is highly convincing and, aside from a minor figure presentation issue, excellent. The biochemical analyses, however, raise concerns that require further clarification and more cautious interpretation.\n\nWe thank this reviewer for their insightful and supportive comments. Please see below for answers to individual points.\n\n1) PARP1 binding and ubiquitination\nThe XL-MS data supporting PARP1 interaction with HECTD3 are strong, but the biochemical validation is less convincing.\nIn Figure 6a, the Coomassie-stained SDS-PAGE shows only a modest shift in PARP1 molecular weight, suggesting attachment of few ubiquitin molecules, whereas the western blot in Supplementary Figure S10c shows distinct higher-molecular-weight. It would be helpful if the authors clarified whether PARP1 is mainly multi-mono-ubiquitinated or polyubiquitinated.\nThe difference in appearance between the Coomassie stained SDS gel and western blot is because longer ubiquitin chains produce stronger immunostaining due to the presence of more epitopes giving the impression that longer ubiquitin chains dominate. Based on reassessment of our data and additional experiments, we now believe that PARP1 becomes mainly multi-mono-ubiquitinated by HECTD3 with some proportion of shorter K48-linked poly-ubiquitin chains (see Supplementary Figs. 10d and 11a). We now report this finding explicitly in the manuscript. Unfortunately, due to the decreased loading of HECTD3 with the ubiquitin K11R mutant (see Supplementary Fig. 9d and further explanation below) we cannot formally distinguish between multi-mono or polyubiquitination by carrying out ubiquitination experiments with the K0 mutant, which would suffer from the same effect.\n\nDo the DOC domain mutants (Y252A, F256A) abolish this higher-molecular-weight species in western blotting, and do they also impair HECTD3 auto-ubiquitination? Furthermore, can the authors exclude that these mutations interfere with E2–E3 charging?\nTo investigate the integrity of DOC domain mutants, we carried out auto-ubiquitination assays with HECTD3 WT and mutant forms (Supplementary Fig. 12c). These experiments showed no effect on E3 activity. Similarly, to confirm that the mutations do not interfere with E2-E3 transthiolation, we have performed E3 loading assays with the same constructs and confirmed that both mutants are still able to be loaded efficiently with ubiquitin (Supplementary Fig. 12d). We have also carried out additional PARP1 ubiquitination assays followed by both, Coomassie staining and western blot (Supplementary Fig. 12b) and found that the mutations in the DOC domain reduce the higher molecular weight species, indicative of reduced substrate binding.\n\nFinally, is the unusual preference for UBE2D E2s over UBE2L3 also observed in PARP1 ubiquitination assays?\nYes. We have carried out additional PARP1 ubiquitination assays that clearly show that the E2 preference is conserved during substrate ubiquitination (see new Supplementary Fig. 11b).\n\n2) Reported substrates not ubiquitinated in vitro\nThe failure to ubiquitinate caspase-8, caspase-9, or STAT3 in vitro is surprising, since these proteins are reported in vivo substrates. Could this reflect a requirement for post-translational modifications or adaptor proteins absent in the assay system? For example, phosphorylation of HECTD3 at Thr157 has been reported as essential for caspase-9 ubiquitination, and MALT1 could act as an adaptor for STAT3 or caspase-8.\nThis is a very good point and we agree, it could be that specific PTMs are required or adaptor proteins that are not present in our in vitro assays. We have now added a sentence to the Discussion to highlight this point.\n\nIn addition, Supplementary Figure 10 shows that HECTD3 auto-ubiquitination is absent in the presence of caspase-8 and caspase-9, which is puzzling since both are apparently no suitable substrates competing with the former activity; an observation that requires further explanation.\nThank you for highlighting this point. The reason auto-ubiquitination of HECTD3 was not detected in the substrate ubiquitination assays was due to the lower concentration of E3 used in this assay set up. We have now repeated substrate ubiquitination assays at higher concentrations (2 µM HECTD3 and 4 µM Substrates) and longer time-points (Supplementary Figs. 10 c and d). Under these conditions we detect strong HECTD3 auto-ubiquitination, which is absent in the reaction with PARP1, where substrate presence suppresses auto-ubiquitination (see new Supplementary Fig. 10).\n\nUltimately, the idea that a constitutively active, and thus uncontrolled, E3 ligase is present in the cytosol is to some extent worrisome. Is it possible that an additional layer of regulation that restricts HECTD3 from futile or even detrimental ubiquitination events is present in vivo. Although a verification of such regulatory mechanisms is most likely beyond the scope of this study, the authors might still want to include some of their thoughts in the discussion.\nThis is a good point. It has been reported in the literature that HECTD3, at least in some cases is regulated at the transcriptional level. The key message from our study is that unlike some HECT family E3s HECTD3 activity is not regulated through autoinhibition or oligomerisation. We agree that we cannot exclude that other mechanisms such as specific PTMs or adaptor proteins play a role in a cellular context. We now comment on this in the Discussion.\n\n3) Ubiquitin chain type\nThe conclusion that HECTD3 primarily catalyzes K11- and K48-linked chains is not fully convincing. Band patterns in Figure 1b and 1c suggest attachment of only a few ubiquitin molecules rather than chain elongation. In UbiCRest assays, residual ubiquitin signals after Cezanne or OTUB1 treatment could reflect priming ubiquitins rather than minor alternative linkages as the authors suggest. Moreover, OTUB1 treatment generates a strong, apparently monoubiquitinated species that persists even with Cezanne co-treatment, possibly pointing to an experimental artifact rather than linkage specificity?\nWe thank this reviewer for highlighting potential issues with the chain linkage specificity of HECTD3. This is particularly relevant with respect to point 4): the interpretation of structural data and possible contribution of ubiquitin K11 to the interface with HECTD3.\nTo address this issue, we have first carried out E2-E3 transthiolation assays with the Ub-K11R mutant to test if this mutation may affect overall activity and hence blur any effect on chain linkage that may exist. These transfer assays show indeed that loading is impaired in the case of the K11R mutant (Supplementary Fig. 9d). It is important to note that this will also affect the use of the K0 mutant that is often used to distinguish between multi-mono and polyubiquitination. In light of these new data, we have reevaluated our interpretation of ubiquitination type and chain linkage specificity and now suggest that HECTD3 mainly acts as a chain initiator forming only short ubiquitin chains.\n\nIn light of this observation, we have removed the data of the UbCRest assays.\n\nFinally, UB-clipping shows that only ~10% of ubiquitin carries two di-glycine remnants, further supporting the idea that HECTD3 primarily mediates attachment of multiple mono-ubiquitination or short chains rather than that of defined long K11 or K48 polyubiquitin chains. Correct?\nApologies, if we didn’t explain the Ub clipping experiment correctly. A single di-glycine remnant signifies that the ubiquitin was part of an unbranched chain or attached to the substrate as mono-ubiquitin, whereas a two di-glycine remnant on ubiquitin signifies that it is a branching point. We agree, that HECTD3 likely mediates primarily multi-mono-ubiquitination and shorter chains. We now say so explicitly in the manuscript.\n\n4) Interpretation of structural data\nThe high-resolution structure of the HECTD3–Ub intermediate suggests that ubiquitin K11 contributes to the interface with the C-lobe. However, it is unclear how much of this interaction is perturbed by K0 or K11R mutants. Could assays characterizing ubiquitin chains be blurred by introduced mutations?\nThis is a very good point. Please see our response to point 3) into which we have discussed this issue.\n\n5) Domain representation\nThe topological description of the D3NA and D3NB domains is difficult to follow and not well presented in Figure 4d. The claim that residues 45–156 form “eight beta-sheets” sounds like a massive domain and should be replaced by “eight b-strands”. Secondary structure assignments are not always convincing (e.g. β-strand 4), and not all strands are labeled in the figure. A clearer illustration with D3NA and D3NB colored differently, and consistent color coding with Supplemental Figure S1, would be better.\nWe thank the reviewer for this helpful suggestion and changed the text accordingly and re-made the figure to more clearly represent the topology (this is now Fig. 3c). We have also corrected the number of beta strands, which indeed contains 7 instead of 8 (also corrected in Supplementary Fig. 1).\n\nReviewer #2 (Remarks to the Author):\nHuber, Rittinger et al. present a comprehensive structure-function analysis of HECTD3 E3 ligase. HECTD3 is one of the 28 human E3 ligases that all contain a C-terminal catalytic HECT domain, but differ in their N-terminal substrate binding and regulatory domains. In cells, HECTD3 was shown to ubiquitinate Caspase-9, PARP1, c-MYC and STAT3. The PDB holds over 200 structures of HECT E3s, as complexes, full-length proteins, or domains. But before now, there were no structures of HECTD3.\n\nMost of the work is a standard set of protein quality and E3 ligase test experiments. The researchers apply SEC-MALS to show HECTD3 is mainly monomeric. Use of the UBIQUENT E2scanTM kit showed most auto-ubiquitination with UBE2D2 and UBE2D3. The researchers do not find indication of auto-inhibition from testing reactivity with Ub-PR. There is not a single ubiquitin chain linkage formed in vitro. Low resolution cryo-EM structures of HECTD3 show the HECTD3 N-terminal domain and inverted T- and L-shaped HECT domain. Use of Ub-PR afforded model building for a HECTD3-Ub intermediate. At the end of the works, the researchers test ubiquitination of the proposed substrates. PARP1 was ubiquitinated in their hands. The researchers performed XL-MS and observed cross-links between PARP1 DBD and HECTD3 DOC domain. Cross-links were also observed between PARP1 NTDs and HECTD3 HECT domain.\n\nSummary: The study provides new knowledge of the E3 ligase HECTD3. The paper is very well-written. The figures in the main text are clear and very aesthetically-pleasing. The novelty comes from the focus on HECTD3. There are not many structures of HECT E3 complexes with substrates so there would be potential for novelty if the authors added a cryo-EM structure of the complex with PARP1. Otherwise, the work mainly extends to HECTD3 what is known from a large body of work on other HECT E3s. Below are specific points to be addressed in order of Figures.\n\nFigure 1. The HECTD3 E2 preference for UBE2D family members is not unique. Many papers in the last years have shown TRIP12 and UBR5 use UBE2D family members.\nThis reviewer is absolutely correct. Other HECT ligases have also been shown to work with UBE2D. We did not intend to claim uniqueness. We only wanted to highlight that it is interesting that the activity of a HECT ligase that functions via an E3~Ub thioester intermediate with the cysteine specific E2 UBE2L3 is so low.\n\nFigure 2. It is unclear from the experiments if HECTD3 is being tested for a preferred activity. It is not clear what percentage of the products are monoubiquitin additions and what percentage are ubiquitin chains. It is also unclear if the auto-ubiquitination of the HECT domain alone is on the same site as FL HECTD3. It is also unclear if this HECT domain reaction proceeds in cis or in trans. Since the researchers cannot conclude if the K11R or K48R mutations have effects beyond linkage they should perform western blotting with linkage-specific antibodies if they wish to conclude preferences. Otherwise, the authors should qualify their conclusions about any linkage specificity for HECTD3.\nWe thank the reviewer for these helpful suggestions and have carried out new experiments to respond to these points.\n\nHere are our answers to individual points raised:\nIt is not clear what percentage of the products are monoubiquitin additions and what percentage are ubiquitin chains.\nUnfortunately, it is not possible to experimentally determine the precise percentage of mono-ubiquitin additions versus poly ubiquitin chains. This is made even harder, given that our new experiments showed that Ub-K11 is important for E2-E3 transthiolation and hence that the ubiquitin K0 mutant cannot be used to assess mono-ubiquitination versus ubiquitin chain formation (please also see our response to reviewer #1, point 3)). However, based on our new experiments we now qualify the text and say that we believe that HECTD3 mainly adds multi-monoubiquitin plus short ubiquitin chains.\n\nIt is also unclear if the auto-ubiquitination of the HECT domain alone is on the same site as FL HECTD3.\nWe have analysed the sites of auto-ubiquitination by mass spec and found that ubiquitination sites in the HECT domain and full-length construct are identical, with additional sites detected in the N-terminal regions of the full-length protein. We prefer not to add these new data to the manuscript as we think that would interrupt the flow of the text.\n\nIt is also unclear if this HECT domain reaction proceeds in cis or in trans.\nTo analyse whether auto-ubiquitination reaction occurs in cis or trans, we performed the following assays: We mixed catalytically inactive GST-tagged HECTD3 HECT domain with wild-type HECT domain and analysed if the former is modified. We found no evidence for modification in trans, even when using western blot detection (anti-Ub antibody). We also performed the assay in the reciprocal manner and similarly detected no convincing sign of trans-ubiquitination of both HECTD3 variants. Nevertheless, given HECTD3’s low tendency for oligomerisation, we cannot fully exclude trans-ubiquitination in a cellular complex where other components may contribute. These experiments are shown below. However, as we do not believe that they are important for the overall message of our paper, we would prefer not to add them to the manuscript.\n\nFigure 3. The researchers should consider removing “highly” from the label since the DOC domain is visible in the maps. It is not clear that data support the conclusion that the DOC domain is “highly” flexible compared to the other domains.\nWe have corrected Figure 3b (now Fig.2b) as requested.\n\nFigure 5e and f. Since the mutations affect E3 loading, the researchers cannot rule out that over multiple cycles the impaired activity would not contribute a lot to the effects on auto-ubiquitination. The conclusions about mechanism cannot be drawn without performing Michaelis-Menten enzyme kinetics and pKa measurements. Those experiments would strengthen the conclusions, but they will not affect the novelty of the paper. So the authors could simply qualify their conclusions.\nWe do not fully understand this point, particularly in relation to pKa measurements. These mutants were generated to understand if their effect was on the E2-E3 transthiolation step or during the subsequent transfer of ubiquitin on to the substrate, the aminolysis step, which requires a catalytic base to deprotonate the target lysine. Given that we assessed which step is affected for each mutant, it remains unclear to us which mechanistic conclusions should be reconsidered.\n\nFigure 6. It is difficult to compare PARP ubiquitination and HECTD3 auto-ubiquitination in this gel system. The bands are too compressed. It would be better to separate by a different gel system or analysis by western blotting.\nWe tested several gel systems to improve separation but none provided a meaningful increase in resolution. However, we believe that the separation achieved is sufficient to demonstrate the absence of any modification on HECTD3 in the presence of PARP1 (please see Supplementary Fig. 10d).\n\nPanel e. The DOC domain Y252A and F256A mutants look like they have general problems. The band below the E2 is lost. There is not FL auto-ubiquitination even at 60 minutes. The authors need experiments showing that the effects are only on substrate binding. The authors should show the cryo-EM data for these residues as an indicator of model accuracy since the interaction with PARP1 is the most novel part of the work.\nThank you for raising this point. To ensure that the DOC domain mutations do not affect the overall stability and activity of HECTD3, we have carried out autoubiquitination and E2-E3 transthiolation assays with the mutants. These new experiments are now shown in Supplementary Fig. 12c and d. These experiments show no effect on autoubiquitination, which however is absent when the correct substrate, in this case PARP1, is present (see Supplementary Fig. 10d).\n\nWe designed the potential interface DOC domain mutants based on sequence conservation and their location on an exposed loop, likely to be accessible to the substrate. They also lie within the proposed substrate binding region of the APC/C DOC domain. These mutants were not designed based on structural observations, and in fact the side-chain density for these residues is poorly defined in the EM map.\n\nSupplementary Figure S1. The statement HECTD3 is highly conserved across species should be reconsidered. The sequence alignment only shows vertebrate species.\nWe thank the reviewer for this over-sight have and have corrected the text as suggested.\n\nSupplementary Figure S2.\nThe researchers clearly demonstrate that HECTD3 is active with UBE2D family members. They should temper their conclusions however. Studying auto-ubiquitination at 60 minutes for the E2 scan could be misleading because there are a lot of steps other than transthiolation occurring by this point in the experiment.\nWe have carried out the E2 scan according to the manufacturer’s instructions. However, to further investigate this point, we have carried out E2-E3 transthiolation assays with UBE2D1 versus UBE2L3 (Supplementary Fig. 2d). These assays clearly show that neither the isolated HECT domain nor full length HECTD3 can efficiently accept ubiquitin from UBE2L3. We hope this resolves any concerns regarding the E2 scan.\n\nThe – and + labels in panel d are confusing. These look to refer to the E2 added but they probably refer to HECTD3. HECTD3 needs to be added to that line.\nWe thank the reviewer for this comment and have modified the figure accordingly (See new Supplementary Fig. 2e)\n\nSupplementary Figure S3. Is it difficult to see HECTD3 auto-ubiquitination in this gel system. The bands are compressed so it is difficult to differentiate effects of the lysine mutants.\nWe agree and therefore have repeated the experiment over a longer time course, which shows the differences between the mutants more clearly. As noted above, we were unfortunately unable to identify a gel system that provided improved band resolution.\nMoreover, our experiments with the K11R mutant showed that this mutant is not suitable for assessing chain linkage specificity, as disrupting Ub K11 affects ubiquitin loading due to contacts with HECTD3 (please see also response to reviewer #1, point 3). Nevertheless, we believe it was important to retain these experiments in the manuscript in order to highlight more generally that these mutants are not always suitable for chain type analysis.\n\nSupplementary Figure S10. The distances to the ubiquitination sites need to be shown if this is to support accuracy of the model.\nAs we have stated in the manuscript on page 11, the predicted HECTD3-PARP1 complex model has low confidence, as indicated by the PAE plot and therefore cannot be used to infer distances to ubiquitination sites. We now recognise that including this model in the manuscript may have given the wrong impression regarding its usefulness and reliability, and have therefore decided to remove it.\n\nSupplementary Figure S11. This figure could be more clear. More depthcue or use of surface representation could be help.\nThank you for this suggestion, we have modified the figure (see new Supplementary Fig. 12e) to better highlight the steric clash at the interface.\n\nResponse to remaining comments from reviewer #2:\nReviewer #2 (Remarks to the Author):\nThis is a solid, clearly wri<en manuscript with well-presented figures. It is a solid addiDon to field, with modest conceptual novelty but the first such data for HECTD3. The new experiments in the revision address many of the reviewers’ earlier concerns. The remaining points could be addressed with experiments noted below, or, alternaDvely, by clarifying the limitaDons of the experiments and conclusions in the text.\nRegarding chain branching, the data indicate that HECTD3 alone does not generate branched chains from ubiquiDn monomers. However, the possibility that HECTD3 could elaborate or branch chains iniDated by another E3 has not been examined. This could be addressed by assays using pre-assembled ubiquiDn chains as substrates to test for branching by HECTD3. If those experiments are not feasible at present, revising the text to clearly state this limitaDon and to acknowledge that future work may reveal a context-dependent role for HECTD3 in chain branching would be necessary.\nTo respond to this point we added the following sentence to the Discussion on page 15:\n“Furthermore, we can’t exclude that HECTD3 might be able itself to branch chains iniDated by other E3s.”\n\nFor the D539A and D855A mutants (Figs. 4e,f aWer the removal of an original figure, and Supp. Fig. 8d), the data do not unambiguously localize the defect to lysine transfer. Given the observed, potenDally pH-sensiDve transthiolaDon defect, the current experiments cannot exclude impairment at the first step to conclude that effects are related to target lysine posiDoning and deprotonaDon influenced by acidic residues in the acDve site. If addiDonal experiments are not feasible, tempering the claims to acknowledge these alternaDve interpretaDons would keep the conclusions well aligned with the data.\nWe have invesDgated the effect of the D539A and D855A mutants on the first step, the transthiolaDon step (see Fig. 4e) which showed that this step is reduced by the mutaDons but not to the same extent that auto-ubiquiDnaDon is reduced (see Fig. 4f), supporDng the noDon that these mutants affect target lysine deprotonaDon. This interpretaDon is further supported by the pH-dependent rescue experiments shown in Supplementary Fig.8d.\nGiven these data we’re not enDrely sure we understand the specific concern by this reviewer.\nIn any case, we have been fairly cauDous in the interpretaDon of our data by staDng that:\n“Thus, both residues likely contribute to catalysis, with opDmal target lysine posiDoning and deprotonaDon depending on the interplay of different acidic side chains in the acDve site.”\nFurthermore, we have changed the sentence in the Discussion page 14\n“Rather, acidic residues (D539 and D855) of the N-and the C-lobe close to the acDve site both play a role in lysine de-protonaDon.”\nTo\n“Rather, acidic residues (D539 and D855) of the N-and the C-lobe close to the acDve site both likely contribute to lysine de-protonaDon”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authorfs did address all my concerns. Congratulations on this lovely piece of work.", "reviewer_2": "This is a solid, clearly written manuscript with well-presented figures. It is a solid addition to field, with modest conceptual novelty but the first such data for HECTD3. The new experiments in the revision address many of the reviewers’ earlier concerns. The remaining points could be addressed with experiments noted below, or, alternatively, by clarifying the limitations of the experiments and conclusions in the text.\n\nRegarding chain branching, the data indicate that HECTD3 alone does not generate branched chains from ubiquitin monomers. However, the possibility that HECTD3 could elaborate or branch chains initiated by another E3 has not been examined. This could be addressed by assays using pre-assembled ubiquitin chains as substrates to test for branching by HECTD3. If those experiments are not feasible at present, revising the text to clearly state this limitation and to acknowledge that future work may reveal a context-dependent role for HECTD3 in chain branching would be necessary.\n\nFor the D539A and D855A mutants (Figs. 4e,f after the removal of an original figure, and Supp. Fig. 8d), the data do not unambiguously localize the defect to lysine transfer. Given the observed, potentially pH-sensitive transthiolation defect, the current experiments cannot exclude impairment at the first step to conclude that effects are related to target lysine positioning and deprotonation influenced by acidic residues in the active site. If additional experiments are not feasible, tempering the claims to acknowledge these alternative interpretations would keep the conclusions well aligned with the data."}, "author_response": ""}]

Decision letter