Structures of ZYG11B-EloB-EloC-substrate complex reveal mechanisms of CRL2^ZYG11B assembly and function

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
ZYG11B is a substrate receptor of the Cullin2-RING E3 ligase (CRL2), mediating the Gly/N-degron pathway and contributing to diverse processes including cell cycle control, protein homeostasis, apoptosis, and innate immunity. While previous studies resolved the structure of its truncated ARM domain, how full-length ZYG11B coordinates substrate engagement and CRL2ZYG11B assembly remains unclear. Here, we present cryo-EM structures of full-length human ZYG11B in complex with the EloB–EloC adaptor and a Gly/N-degron peptide, revealing a seahorse-like architecture with distinct interfaces for adaptor and substrate binding. Unexpectedly, ZYG11B adopts both monomeric and dimeric assemblies, with the dimer stabilizing two substrate-binding sites in opposite orientations. Functional assays demonstrate that interfaces mediating adaptor recruitment, substrate binding, and dimerization are essential for substrate degradation, suggesting a dynamic mechanism involving both assembly states. These findings provide a structural framework for understanding CRL2ZYG11B-mediated ubiquitination and offer mechanistic insights that may inform the rational design of ZYG11B-based applications.

Introduction
The ubiquitin-proteasome system (UPS), together with autophagy, constitutes the two major cellular protein quality control pathways1-4. UPS-mediated degradation is initiated by a hierarchical enzymatic cascade involving E1 (activation), E2 (conjugation), and E3 (ligation) enzymes, which catalyze the ubiquitination of target proteins destined for degradation by the 26S proteasome5-8. Substrate specificity is conferred by E3 ligases, which function as either single polypeptides or multi-subunit complexes. Typically, E3s comprise a substrate-binding module that recognizes specific degradation motifs (degrons) and a catalytic module that transfers ubiquitin from E2 to substrates9-11.

Various types of degrons have been identified, including N-degrons (the well-known N-end rule pathway), internal degrons, and C-degrons, often exposed upon enzymic cleavage or posttranslational modification9,12,13. To accommodate this diversity, E3s have evolved distinct recognition mechanisms. Based on structural and catalytic features, E3s are broadly categorized into RING (Really Interesting New Gene), HECT (Homologous to E6AP C-terminus), and RBR (RING-between-RING) families. While RING E3s catalyze direct ubiquitin transfer from E2 to substrate, HECT and RBR E3s form a transient E3-ubiquitin thioester intermediate prior to ligation10,11,14. Among E3s, Cullin-RING ligases (CRLs) represent the largest subfamily. CRLs are assembled on one of eight Cullin scaffolds (Cul1-3, Cul4A/B, Cul5, Cul7, Cul9), and typically consist of a RING protein (Rbx1 or Rbx2), an adaptor, and a substrate receptor5,14-19. This modular architecture enables CRLs to target a broad spectrum of substrates, accounting for up to 20% of UPS-mediated proteolysis14-16.

Recent study identified ZYG11B and ZER1—two members of the ZYG family—as substrate receptors that recognize the Gly/N-degron, a previously uncharacterized degron type defined by an N-terminal glycine residue, and recruit Cul2 to form the CRL2ZYG11B and CRL2ZER1 E3 ligases, respectively20. In metazoan cells, Gly/N-degrons are typically underrepresented in the proteome and become exposed upon caspase cleavage or defective N-myristoylation, suggesting critical roles for ZYG11B and ZER1 in apoptotic signaling and membrane targeting20,21. In addition, the ZYG11B-mediated Gly/N-degron pathway could be utilized during viral infections22-25. For instance, the NLRP1 inflammasome is activated when ZYG11B recognizes a Gly/N-degron signal of NLRP1 exposed by enteroviral 3C protease cleavage22, while SARS-CoV-2 ORF10 protein binds ZYG11B in a Gly/N-degron-dependent manner to influence viral infection through an inclusive mechanism23-25. Notably, ZYG11B can also promote degradation of substrates lacking canonical degrons, such as the cell cycle regulator cyclin B1, suggesting additional layers of substrate recognition26.

ZYG11B contains three domains: an N-terminal von Hippel-Lindau (VHL) box, a central Leucine- Rich Repeat (LRR) domain, and a C-terminal Armadillo Repeat (ARM) domain. Despite previous structural determination of a truncated ARM domain covalently fused to Gly/N degron peptides27-29, how full-length ZYG11B coordinates substrate binding and adaptor proteins to assemble into the functional CRL2ZYG11B E3 ligase remains unclear.

Here, we present cryo-electron microscopy (cryo-EM) structures of full length human ZYG11B in complex with a substrate peptide and the EloB-EloC adaptor complex, and further model the intact CRL2ZYG11B holoenzyme. Complemented by biochemical experiments and cell-based assays, these findings demonstrate that ZYG11B mediates both monomeric and dimeric assemblies of the CRL2ZYG11B complex, with dimerization being crucial for efficient substrate ubiquitination and degradation.

Results
ZYG11B mediates both monomeric and dimeric assemblies

To elucidate how ZYG11B recruits EloB-EloC adaptor complex and engages substrates, we sought to determine the cryo-EM structures of ZYG11B-EloB-EloC complex bound to the degron peptide, both in the absence and presence of the Cul2-Rbx1 module. To this end, full-length ZYG11B was co-expressed and purified together with EloB and EloC to form the ZYG11B-EloB-EloC core complex, which was subsequently incubated with Cul2-Rbx1 to form the ZYG11B-EloB-EloC-Cul2-Rbx1 holoenzyme complex. Both samples exhibit favorable biochemical behavior and good purity, as assessed by size-exclusion chromatography and SDS-PAGE (Supplementary Fig. 1a). Unexpectedly, two-dimensional classification of the cryo-EM data reveals that both complexes adopt not only monomeric but also dimeric assemblies (Supplementary Fig.1b). Analytical ultracentrifugation (AUC) assays further confirm the coexistence of monomeric and dimeric species in solution, regardless of the expression system or buffer ionic strength (Supplementary Fig.1c), suggesting that both assembly states are intrinsic properties of ZYG11B-containing complexes. Given that some CRL substrate receptors are known to mediate oligomerization30-35, we speculated that the dimerization process was mediated by ZYG11B itself. In support of this hypothesis, co-immunoprecipitation assays using differentially tagged ZYG11B constructs demonstrate self-association in cells (Supplementary Fig.1d), consistent with the previous report36. These findings indicate that ZYG11B can form homodimers both in vitro and in cells.

We next reconstructed near-atomic cryo-EM structures of the monomeric and dimeric ZYG11B-EloB-EloC core complexes bound to a Gly/N-degron peptide (GYIND) derived from the SARS-CoV-2 ORF10 protein, at global resolutions of 3.37 Å and 3.27 Å, respectively (Fig. 1, Supplementary Fig.2, 3 and Table 1). The monomeric complex contains one copy of ZYG11B-EloB-EloC-substrate at a 1:1:1:1 stoichiometry (Fig. 1a, b), while the dimeric assembly is composed of two such units arranged symmetrically (Fig. 1c, d). These structures clearly show that ZYG11B mediates dimer formation, in line with the biochemical and cellular data. Structural alignments indicate that the core module adopts a similar conformation in the monomer and in each unit of the dimer (Supplementary Fig.4a). Structure of ZYG11B also superposes well with its AlphaFold model (Cα RMSD = 1.56 Å), indicating the reliability of AlphaFold predictions for other ZYG11 family members (Supplementary Fig.4b-d).

Structure of full-length ZYG11B

The full-length ZYG11B folds into an overall seahorse-shaped architecture, with the N-terminal VHL box and the central LRR domain forming the head region, and the C-terminal ARM domain constituting the body and tail regions (Fig. 2a, b). The LRR and ARM domains pack tightly with each other through intramolecular hydrogen bonding and hydrophobic interactions, thereby stabilizing the overall structure (Fig. 2c). The VHL box contains a canonical BC box that spans the first N terminal helix of ZYG11B, followed by a Cullin box (Fig. 2d). Notably, ZYG11B features an unusually long and extended loop connecting these two motifs, which distinguishes it from other VHL box-containing proteins (Fig. 2d and Supplementary Fig. 5a)37. Structural similarity searches for the ZYG11B VHL box using Dali server against the Alphafold Database revealed that this extended loop is unique to the ZYG11 protein family, with the exception of ZYG11 from Caenorhabditis elegans (Supplementary Fig. 5b). The LRR domain constitutes of ten α-helix/β-strand repeats and adopts a curved solenoid conformation. In this configuration, ten α-helices align along the convex surface, while ten parallel β-strands define the concave groove (Fig. 2e). The ARM domain is composed entirely of α-helices and interconnecting loops that fall into nine ARM repeats. The α-helices maintain the overall architecture, while the loops introduce variations in inter-repeat angles and orientations (Fig. 2f). Particularly, a long flexible linker between ARM7 and ARM8 prevents the whole ARM domain from folding into a continuous superhelical cylindrical structure, allowing ARM8 and ARM9 to swing back and shape the substrate binding pocket. This configuration is observed in our full-length structure and is consistent with previously reported crystal structures of the truncated ARM domain lacking ARM1-3 (Fig. 2f) 29. Meanwhile, structural alignment also reveals a subtle conformational difference in ARM4 relative to our full-length structure, which may reflect the effects of protein truncation or crystal packing (Supplementary Fig. 5c).

Structural basis of ZYG11B recruiting EloB-EloC and substrate

In the complex, EloB-EloC is recruited to the N-terminal VHL box of ZYG11B, while the twisted C-terminal ARM domain folds back to engage EloB, together resembling a bent ‘Mobius strip’ like conformation (Fig. 3a). ZYG11B interacts with EloB-EloC through three distinct interfaces involving extensive hydrogen bonds and hydrophobic contacts (Fig. 3b). The first interface (interface 1) is located at the VHL box, where EloC tightly associates with the BC box of ZYG11B, forming a large contact area. This interaction mode resembles those observed in other CRL2 substrate receptors, such as the von Hippel-Lindau factor and FEM1B protein (Supplementary Fig. 5d)38. Specifically, L18 of the BC box inserts into a hydrophobic pocket on EloC formed by residues V73, Y76, F93, I95, L103, A107, and L110 (Fig. 3c), while L104 of EloC fits into a hydrophobic cavity on the opposite side, composed of ZYG11B residues L19, C22, L23, L26, F50, P51, V54, and L58 (Fig. 3d). Additionally, two smaller flanking hydrophobic surfaces contribute to this interface: one involves BC box residue I21 and EloC residues Y76, Y79, F93, and I95; the other includes BC box residues L26, F33, E46, and F50 together with EloC residues L101 and M105 (Fig. 3e). Hydrogen bonds between A13, S14, Y16, L18 of the BC box and N85, Y76 of EloC further stabilize the interface (Fig. 3f). Two additional interaction surfaces, referred to as interface 2 and interface 3, further stabilize the complex. Interface 2 comprises residues A397 and R436 of the ARM3 domain contacting EloC residue S87, while interface 3 involves ARM9 residues R728 and H729 electrostatically interacting with EloB residues E59—an interaction not observed in other CRL2 substrate receptors (Fig. 3g, h).

The degron peptide binds snugly within the groove of ARM domain, with its N-terminal glycine residue inserting into a substrate-binding pocket and its C-terminus exposed to solvent (Fig. 3a). This binding mode is consistent with previous crystal structures of truncated ARM domains fused to degron peptides (Supplementary Fig. 5c)27-29. In our full-length structures, the first three residues of the degron are firmly anchored within the binding cavity through a main-chain mediated hydrogen-bonding network involving ZYG11B residues W522, N523, D526, N567, E570, and A647 (Fig. 3i). The side chains of Y2 and I3 from the degron form additional hydrogen-bonding and hydrophobic interactions with R649 and W522, respectively (Fig. 3j). Although the fourth residues of the degron remain buried within the pocket, no specific interactions with ZYG11B were observed in our structures (Fig. 3i).

To validate the functional importance of these interactions, we conducted mutagenesis studies and evaluated the substrate stability of the degron-fused proteins using the global protein stability system (GPS) in vivo (Fig. 3k)20. The degron sequence was derived from DBNDD2, a known endogenous substrate of ZYG11B. In this assay, HEK293T cells were stably transfected with a vector encoding ubiquitin-(N-glycine-degron)-EGFP-P2A-DsRed. Upon translation, P2A-mediated cleavage produces separate ubiquitin-degron-EGFP and DsRed proteins. Following deubiquitination, the degron-EGFP is recognized by ZYG11B and directed to proteasome degradation, and the EGFP/DsRed ratio can reflect the stability of the substrate and the activity of ZYG11B. As expected, expression of wild-type ZYG11B remarkably reduced the EGFP/DsRed ratio through promoting degradation of the degron-fusion EGFP (Fig. 3l). In contrast, the W552A mutation within the substrate-binding pocket failed to promote degradation, whereas the empty vector condition exhibited slightly increased substrate stability than mutants, probably due to a dominant-negative effect. Moreover, mutants disrupting the EloC-interacting interface (mutant1: L18S; mutant2: P47A/V49A/F50A/P51A) retained high EGFP/DsRed ratios compared to wild-type ZYG11B, indicating impaired activity (Fig. 3l). Collectively, these results highlight the vital roles of specific ZYG11B residues in recruiting EloB-EloC and recognizing substrates.

Structural details of ZYG11B-mediated dimerization

The dimeric assembly comprises two copies of the ZYG11B-EloB-EloC-substrate complex, forming a 2:2:2:2 stoichiometry with C2 symmetry. Within the dimer, the two ZYG11B molecules are arranged back-to-back, each recruiting an EloB-EloC complex and capturing a degron peptide, with the peptide C-termini oriented in opposite directions (Fig. 4a and 1c). All three domains of ZYG11B—the VHL box, LRR domain, and ARM domain—contribute to the formation of an extensive, tightly packed dimer interface, spanning approximately 1358 Å2 (Fig. 4b). Specifically, Q38 from the protruding loop of the VHL box forms hydrogen bonds with E460, Q499, and Q503 of the opposing ARM domain (Fig. 4c). A second hydrogen-bonding network is organized by I93, S94, N116, and D118 of the LRR domain and N420 and H421 of the opposing ARM domain (Fig. 4d). Additionally, L150 from the LRR domain inserts into a hydrophobic pocket formed by L377, L381, and H421 of the opposing ARM domain, and further establishes a hydrogen bond between the main chain of L150 and the side chain of R342 (Fig. 4e). Finally, the two LRR domains also interact directly at two sites: one featuring a hydrogen bond between D152 and K248, together with hydrophobic contacts between Y175 and the backbone and side chains of Q249 and F250; and a second site involving a crosswise hydrogen-bond network formed by K220, L222, and K223 (Fig. 4f, g). Owing to the two-fold symmetry, these interactions are reciprocally mirrored on the opposite side of the dimer. Together, these interlocking interactions robustly stabilize the dimeric architecture.

To further investigate the full holoenzyme assembly, we reconstructed a low-resolution cryo-EM map of the hexamer complex containing ZYG11B-ELoB-EloC-Cul2-Rbx1 and the degron peptide (Supplementary Fig. 1b). By aligning our ZYG11B-ELoB-EloC-degron structure with the previously reported VHL-ELoB-EloC-Cul2-Rbx1 structure (PDB: 5N4W, [https://doi.org/10.2210/pdb5N4W/pdb]) via the VHL-EloB-EloC subunit, we built a composite model of the intact CRL2ZYG11B E3 ligase (Supplementary Fig. 6a). This model fits well into the cryo-EM map and positions the C-terminus of the degron peptide towards the catalytic subunit Rbx1, thereby providing a direct spatial basis for coupling substrate recognition with ubiquitin transfer (Supplementary Fig. 6a, b). We further built a dimeric model of the holoenzyme applying the same C2 symmetry observed in the ZYG11B-ELoB-EloC-degron dimer (Supplementary Fig. 6c). The resulting model exhibits no steric clashes and aligns with the 2D class averages (Supplementary Fig. 1b). Together, these analyses support the structural plausibility and biological relevance of a dimeric CRL2ZYG11B E3 ligase assembly.

To evaluate the functional importance of the dimeric assembly, we first established the in vitro ubiquitination assay to assess enzymatic activity. Given the extensive buried surface area of the dimer interface, a potential dimer-disrupting mutant was designed, incorporating combined substitutions containing L150S/D152S/R153S/K223A/F418A/P419A/N420A. The substrate contains an N-terminal ZYG11B-recognized degron peptide (GYFQRGK) which is followed by a long GS linker and a C-terminal GFP-FLAG tag 29. As shown in the time-course biochemical assay (Supplementary Fig. 7a, b), wild-type CRL2ZYG11B promoted detectable mono- and di-ubiquitination of the substrate within 5 min, whereas the dimer-disrupting mutant showed weaker ubiquitination at all examined time points. Peptide-competition and enzyme concentration–gradient experiments further confirmed that substrate ubiquitination was dependent on CRL2ZYG11B E3 ligase activity (Supplementary Fig.7c, d). Moreover, in subsequent in vivo GPS assays, this mutant significantly impaired the degradation of degron-fused proteins compared with wild-type ZYG11B and other insufficient mutations (Fig. 4h and Supplementary Fig. 8a, b). These results indicate that dimerization of CRL2ZYG11B is critical for its E3 ligase activity and related biological functions.

Methods
Plasmid construction

The genes encoding the CRL2ZYG11B complex members, including full-length ZYG11B, EloB, EloC, Cul2, and Rbx1, were amplified from human complementary DNA (cDNA) using standard PCR methods. The ZYG11B coding sequence was cloned into a modified pFastBac Dual vector to express ZYG11B-TEV-GFP-6×His, or into pRSFDuet to express a His-SUMO-tagged ZYG11B. Human EloB and EloC were cloned into the modified pFastBac Dual vector or pACYC Duet vector at the first expression site (BamHI, HindIII) with a TEV-cleavable N-terminal His tag and at the second expression site (XhoI, KpnI) without a tag, respectively. The human Cul2 and Rbx1 genes were also cloned into the pFastBac Dual vector at cloning sites 1 (with an N-terminal His-tev tag) and 2 (without tag), respectively.

We made pCDH-blasticidine-ubiquitin-(N-degron)-EFGP-P2A-DsRED constructs for GPS-reporter cells using the CloneExpress one-step cloning kit (Vazyme). The N-terminal peptides of DBNDD2 were cloned into the GPS-reporter vector. Wild-type and mutant ZYG11B were cloned into the pCDH-cmv-puro vector with an N-terminal Flag tag.

For in vitro ubiquitination assays, expression constructs for all recombinant proteins were generated as follows. Ubiquitin (Ubi), UBE2R, and the substrate fusion GYFQRGK-(GS)20-GFP-FLAG were cloned into a modified pRSF vector encoding an N-terminal His–SUMO tag, generating pRSF-His-SUMO-Ubi, pRSF-His-SUMO-UBE2R, and pRSF-His-SUMO-GYFQRGK-(GS)20-GFP-FLAG. UBE2D3 was cloned into pET-28a with an N-terminal His tag followed by a TEV protease cleavage site (pET28a-His-TEV-UBE2D3). UBA1 was cloned into pFastDual with an N-terminal His–TEV tag (pFastDual-His-TEV-UBA1). All constructs were verified by Sanger sequencing.

Protein expression and complex purification

The ZYG11B-EloB-EloC ternary complex was co-expressed in Sf9 cells (Thermo Fisher Scientific, cat. no. 11496015) by co-infection with P3 baculoviruses encoding ZYG11B and the EloB-EloC bicistron at a 2:1 ratio. Recombinant baculoviruses encoding the CUL2–Rbx1 complex and UBA1 were amplified in Sf9 insect cells. Sf9 cells at a density of 2.0-2.5 × 106 cells ml−1 were infected with the indicated baculoviruses and cultured for 72 h at 27 °C. Cells were harvested by centrifugation (2,000 × g, 10 min). Expression vectors encoding ZYG11B-EloB-EloC, ubiquitin, UBE2R, UBE2D3, and the substrate GYFQRGK-GFP-FLAG were transformed into E. coli BL21(DE3) cells. Protein expression was induced with 0.3 mM IPTG and cultures were grown overnight at 16 °C.

For purification of ZYG11B-EloB-EloC proteins, cell pellets were resuspended and lysed by ultrasonication in lysis buffer containing 50 mM Tris-HCI (pH 7.5), 200 mM or 500 mM NaCl, 10 mM imidazole, 5% glycerol, 2 mM β-mercaptoethanol (β-ME), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The recombinant proteins were initially purified by Ni2+ affinity chromatography. The supernatant was loaded onto the Ni2+ column; then the protein was washed with 50 mM imidazole and eluted with 500 mM imidazole in low salt (50 mM NaCl) or high salt (300 mM NaCl) buffer, respectively. The eluate was treated with TEV and ULP1 protease digestion overnight and loaded onto a Ni2+ column to remove the tags. The protein was then subjected to size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare), equilibrated with low-salt buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 1 mM DTT) or high-salt buffer (20 mM HEPES, pH 7.5, 300 mM KCl, 1 mM DTT). Prior to cryo-EM grid preparation, the synthetic substrate peptide GYIND was incubated with the ZYG11B-EloB-EloC complex on ice for 30 min at a 10:1 (peptide: complex) molar ratio.

The Cul2-Rbx1 complex was purified using a similar protocol, with minor modifications. The final purification step involved gel filtration in 20 mM HEPES (pH 7.5), 150 mM KCl, and 1 mM DTT. The Cul2-Rbx1-ZYG11B-EloB-EloC pentamer complex was assembled by mixing the purified components and subjected to size-exclusion chromatography in 20 mM HEPES (pH 7.5), 150 mM KCl, and 1 mM DTT. The ZYG11B-EloB-EloC-Cul2-Rbx1 complex was supplemented with the synthetic substrate peptide GYIND at a 10:1 (peptide: complex) molar ratio and incubated on ice for 30 min before cryo-EM grid preparation. The neddylated Cul2Δ-Rbx1 (Cul2Δ: Δ117-134) protein used in in vitro ubiquitination assays was a kind gift from Dr. Chao Xu at University of Science and Technology of China47.

For proteins used in in vitro ubiquitination assays, including ubiquitin, UBA1, UBE2R, UBE2D3, and the substrate GYFQRGK-GFP-FLAG, recombinant proteins were initially purified by Ni2+-affinity chromatography. Affinity tags were removed by overnight digestion with TEV protease or ULP1, followed by a second round of Ni2+-affinity chromatography to remove the cleaved tags and proteases. The final proteins were stored in buffer containing 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% (v/v) glycerol.

Analytical ultracentrifugation (AUC) assays

Sedimentation velocity analytical ultracentrifugation (SV-AUC) experiments were performed using a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter, Brea, CA), equipped with an AN-60Ti rotor (4-holes). Prior to the experiments, an additional purification step of the ZYG11B-EloB-EloC complex was performed using size-exclusion chromatography in low-salt buffer (20 mM HEPES, pH 7.5, and 50 mM KCl) or high-salt buffer (20 mM HEPES, pH 7.5, and 300 mM KCl). Samples were loaded with 380 μL of ZYG11B-EloB-EloC at a concentration of about 1mg/ml and 400 μL of the same purification buffer. SV-AUC experiments were carried out at 20°C and 70,000 × g using a continuous scan mode with a radial spacing of 0.003 cm. Scans were collected in 3 min intervals at 280 nm. The fitting of absorbance versus cell radius data was performed using SEDFIT software and continuous sedimentation coefficient distribution c(s) model, covering a range of 0-20 S.

Cryo-EM specimen preparation and imaging

Three microliters (3 μL) of purified ZYG11B-EloB-EloC with the substrate peptide (1 mg/ml) were applied to glow-discharged Quantifoil R1.2/1.3 Cu grids. The grids were incubated for 2 s, blotted for 6 s at 4 ℃ and 100% humidity, and then plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Data collection of all the cryo-EM datasets was performed on the 200 kV Talos Arctica electron microscope (FEI) equipped with K2 summit camera (Gatan). All cryo-EM micrographs were collected automatically under super-resolution mode, yielding an image stack with a pixel size of 0.8 Å (×165,000). Images were recorded at a defocus range of -1.2 μm to -1.8 μm, with a total electron dose of 60 e− Å −2 over 32 movie frames.

Image processing

A total of 1987 micrographs were collected for ZYG11B-EloB-EloC-substrate dataset. Movies frames were aligned using Patch Motion Correction, and CTF parameters were estimated using Patch CTF Estimation in cryoSPARC. In total, 690,827 particles were picked and extracted. Multiple rounds of 2D classification yielded 347,626 particles with clear features, which were subjected to Ab initio reconstruction. After Ab initio reconstruction, two well-defined classes corresponding to monomeric and dimeric ZYG11B-EloB-EloC complexes were selected for further processing. For the monomer dataset, non-uniform refinement followed by local refinement generated a 3.37 Å map from 99,806 particles. For the dimer dataset, two rounds of non-uniform refinement with C2 symmetry produced a 3.27 Å map from 100,431 particles.

Model building and refinement

The sharpened maps were generated in cryoSPARC. An initial model of the ZYG11B-EloB-EloC-substrate complex was built based on the AlphaFold-predicted structure of ZYG11B (AF-Q9C0D3-F1), together with the crystal structure of EloB-EloC (PDB ID: 5N4W[https://doi.org/10.2210/pdb5N4W/pdb]) and the ORF10 peptide (PDB ID: 7XV7 [https://doi.org/10.2210/pdb7XV7/pdb]). The model was fit into the cryo-EM maps using UCSF Chimera and further manually adjusted in Coot. The resulting model was refined in PHENIX by real-space refinement with secondary-structure restraints. The final models were subjected to multiple rounds of real-space refinement in PHENIX and manual adjustment in Coot until convergence, with no further improvement observed. Model stereochemistry was validated using the comprehensive cryo-EM validation tools in PHENIX. Refinement statistics are summarized in Supplementary Table 1. Structural figures were prepared using PyMOL or ChimeraX.

Cell culture and Lentivirus packaging

Human HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Biological Industries, Cat. No. 01-052-1ACS) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Cat. No. 04-001-1ACS) and 1% penicillin-streptomycin (HyClone) at 37 °C in a 5% CO₂ incubator. For lentiviral packaging, transfer vector constructs (pCDH-ub-DBNDD2 peptide-EGFP-P2A-DsRed and pCDH-ZYG11B-WT or mutant), along with packaging plasmids psPAX2 and pCMV-VSV-G, were co-transfected into HEK293T cells. Forty-eight hours post-transfection, the lentivirus was harvested from the supernatant and filtered through a 0.45 μm filter (Millpore).

Global protein stability assay

The GPS assay using the Ub-DBNDD2 peptide GPS reporter cell line was previously described (Xiaojie Yan et al., 2022). Briefly, the 19-mer DBNDD2 peptide was encoded as an oligonucleotide and cloned into the GPS vector. To generate GPS reporter cells, the GPS vector was packaged into lentivirus and transduced into HEK293T cells, followed by selection with blasticidin (30 μg/mL). To assess ZYG11B activity, lentiviral vectors encoding wild-type or mutant ZYG11B with an N-terminal Flag tag were packaged and transduced into GPS reporter cells, followed by selection with puromycin (10 μg/mL). Fluorescence signals of EGFP and DsRed were measured by flow cytometry using an LSR Fortessa instrument (Becton Dickinson). For the flow cytometry gating strategy, cells were first gated on FSC-A and SSC-A to exclude debris and doublets. DsRed-positive cells were then selected as events with DsRed fluorescence intensity > 103, representing cells carrying the lentivirally integrated GPS reporters. GFP/DsRed fluorescence ratios were analyzed with FlowJo software and used to quantify the stability of the GFP-fused substrate. In the resulting distributions, the y-axis represents the normalized frequency of cells. Cells were also analyzed by Western blotting. The following primary antibodies were used: anti-FLAG antibody (mouse monoclonal, ABclonal, Cat# AE005; 1:2000), anti-GAPDH antibody (mouse monoclonal, ABclonal, Cat# AC033; 1:5000), and anti-β-actin antibody (mouse monoclonal, ABclonal, Cat# AC004; 1:5000). HRP-conjugated goat anti-mouse IgG (H+L) (ABclonal, Cat# AS003; 1:10000) was used as the secondary antibody.

In vitro ubiquitination assays

For in vitro ubiquitination assays, UBA1 was used as E1 enzyme, and two E2 enzymes (UBE2R1 and UBE2D3) combination were used to enhance efficiency according to previous reports48. The assays were performed at 37 °C in reaction buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM MgCl₂) containing 1 μM UBA1, 1 μM UBE2R1, 1 μM UBE2D3, 1 μM neddylated Cul2Δ-Rbx1, indicated amounts of wild-type ZYG11B-EloB-EloC or mutants, 1 μM substrate GYFQRGK-GFP-FLAG, and 100 μM ubiquitin. 5 mM ATP was added to the mixture to initiate the reaction. For competition experiments, degron substrate peptide (GYFQRGK) was added to the mixture at indicated concentrations. Reaction systems were mixed with SDS-PAGE loading buffer after indicated time and boiled for 10 minutes to terminate the reaction. Ubiquitinated products were detected by immunoblotting using an anti-GFP antibody (mouse monoclonal, ABclonal, Cat# AE012; 1:2000 dilution), followed by an HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (ABclonal, Cat# AS003; 1:10000 dilution).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Lin et al. reports very nice cryoEM-derived structures of full-length ZYG11B in complex with other components of this interesting E3 ligase plus a substrate-derived peptide. Prior structures used fragments of ZYG11B. While the current manuscript presents compelling structural biology, functional assays to support implications of the structural findings are limited or have caveats. Below are my specific comments, in approximate order of importance.\n1) Figure 4g: I commend the authors for providing the immunoblots to show relative levels of the ZYG11B variants in cells. My concern is that the “dimer surface mutant” was at significantly lower levels than the WT protein. What would the data/plot look like if WT levels were matched with the mutant?\nEven if ZYG11B variants were matched in levels by blot, their cellular localizations or folding quality could be different, especially given the high number of amino acid substitutions for the dimer surface mutant and overexpression. CRL substrate recognition subunits have long been known to not fold properly in the absence of their binding partners and specific chaperones (Feldman et al., PMID:10635329; Taipale et al., PMID:25036637). The authors would need to demonstrate both equal levels plus equal functionality in some way, such as equal co-IP of EloB/C.\nAn alternative is to use biochemistry to investigate E3 activity (see comment #2).\n2) Supplemental Figure 5 shows nice data for the presumed full E3 complex: CUL2/Rbx1/EloB/EloC/ZYG11B. I write “presumed” because I have not seen any report of reconstituted activity for this E3, with a peptide substrate for example. If the authors could develop such an assay, it would greatly enhance the current manuscript and allow for more controlled functional tests of their structure-based hypotheses. I suggest using the DBNDD2-derived peptide with added lysine residues and a tracer near the C-terminus. See Hickey et al. (PMID:38177675) for an analogous assay for CRL2-KLHDC2. Can the dimer surface mutant be purified? If so, is it less active than the dimer? Only about 2-fold less active? Does it bind EloB/C and substrate peptide? Other mutants could also be studied, but I think the implications of dimerization are most pressing—and novel—for the current manuscript.\nThere are also some interesting opportunities with a biochemical activity assay and purified mixed ZYG11B dimers (would require two different tags). For example, would a dimer with two (non-mutated) active sites be only twice as active as a dimer with only one active site?\nCombined, these additional studies may suggest that the main reason ZYG11B dimerizes is for enhanced protein stability. More interesting findings are also possible, though from the current manuscript I only see evidence for dimerization creating two identical substrate-binding pockets that could likely function in monomers. The authors even present a model in which monomers and dimers contribute to E3 activity. Biochemical activity assays could provide significant resolution.\n3) The manuscript discussion mentions “a valuable framework for the development of ZYG11B-based PROTACs”. I did not find obvious ways that the manuscript would aid in PROTAC design. Again, I see two identical substrate-binding pockets consistent with previous reports. Does the dimer interface create another potentially ligandable site? For KLHDC2, tetramerization might explain relatively weak PROTACs (or ligands for PROTACs) if the PROTACs/ligands are not capable of disrupting the tetramer, as the peptides do. From the current manuscript, it does not appear that ZYG11B dimerization would present a problem or advantage for PROTACs. Please elaborate if there are specific ideas.\n4) Supplemental Figure 1 (and methods): It’s not clear to me which conditions had the substrate peptide. Parts A through C of the figure? Did the authors perform experiments to determine whether the substrate peptide influences the monomer-dimer state?\nMinor comments:\n5) Methods, line 275: please provide more detail on the pCDH-puro vector. CMV promoter? There are many versions of this vector. The promoter in the plasmid matters since the level of protein overexpression in cells can have important implications.\n6) Methods, line 266: “C-terminal HIS-GFP-TEV tag” is confusing to me. It is best to list proteins in N to C order. So, was it ZYG11B-TEV-GFP-6HIS, leaving no tag behind after cleavage?\n7) Figure 5: The proteasome cartoon would be better if made simpler (just one shape) instead of appearing as 10 stacked hexamers, which is not accurate. Or, perhaps Nature Publishing can provide a better proteasome cartoon that more accurately depicts the lid, base, and CP parts of the proteasome. Also, the color used for Ub makes it difficult to see above the background.", "reviewer_2": "This manuscript presents structural and functional analyses of the human CRL2^ZYG11B E3 ubiquitin ligase, focusing on how ZYG11B engages substrates bearing Gly/N-degrons. Using cryo-electron microscopy, the authors report structures of full-length ZYG11B bound to the EloB–EloC adaptor complex and a Gly/N-degron peptide in both monomeric and dimeric assemblies. Complementary cell-based assays support the notion that disrupting substrate, adaptor, or dimerization interfaces impairs substrate degradation. While the study offers some new observations, there are several major concerns that should be addressed to clarify the novelty and rigor of the findings.\nFirst, a closely related study was posted on bioRxiv in 2024 and is only briefly mentioned in the Discussion. The authors do not adequately acknowledge that the previously reported structures overlap significantly with those presented here. This raises concerns about the originality of the work and diminishes its novelty. The manuscript should provide a more transparent comparison to the bioRxiv study and clearly articulate what is uniquely contributed here.\nSecond, the reported ZYG11B dimer represents a potential novel finding not described in the bioRxiv paper. However, the biological relevance of this dimer is not convincingly established. Specifically, the manuscript lacks structural data of the full CRL2^ZYG11B complex including Cul2, which would be necessary to demonstrate that the dimeric form is retained upon assembly with the full E3 ligase machinery. The only supporting evidence—weak density corresponding to Cul2 in 2D class averages—is not sufficient. Stronger experimental validation, such as reconstruction of the dimeric complex with Cul2-Rbx1, is needed to substantiate this claim.\nThird, the structural data are not presented with sufficient clarity or transparency to allow readers to evaluate the quality of the reconstructions. The reported resolutions (~4 Å) are modest and may not support unambiguous side chain assignments, yet the manuscript contains detailed interaction descriptions (e.g., hydrogen bonding networks) that may go beyond what is justified by the data. With the exception of Fig. 1, cryo-EM density maps are not shown in the main figures. Furthermore, figures such as 2C, 3a, 3c–e, 3j, and 4e–f appear to use surface renderings derived from atomic models, not the experimental maps themselves, which could mislead readers about the level of structural detail actually supported by the data. The authors state that AlphaFold models were used for initial model building, but it remains unclear to what extent the final structural conclusions rely on experimental evidence versus predicted models.\nFourth, the GPS degradation assays are insufficiently described and potentially misleading. The presentation of the data lacks normalization details; the Y-axis scale and its relation to population-level distributions are unclear. Mutant constructs show effects beyond the vector control, which suggests potential dominant-negative behavior that is not explored or acknowledged. The influence of variable expression levels, as seen in Western blots, is not discussed. Moreover, the choice of mutations—particularly the combination of L150S/D152S/R153S/K223A/F418A/P419A/N420A—seems arbitrary and is the only construct used to support the functional relevance of dimerization. Additional mutants or rescue experiments would be helpful to strengthen this conclusion.\nMinor points:\nLine 101: The term “electron density” is not appropriate in the context of cryo-EM and should be replaced with “electrostatic potential map” or “Coulomb potential.”\nThe title is somewhat misleading; the study focuses on the ZYG11B–EloB–EloC complex and substrate recognition. The involvement of the full CRL2 ligase (including Cul2 and Rbx1) is limited and not structurally characterized in depth.\nIn summary, while the manuscript contains potentially interesting observations, the novelty relative to prior work, the interpretation of the structural data, and the functional validation all require clarification or strengthening to meet the standards of a high-impact journal."}, "author_response": "Overall Response:\nWe sincerely thank the reviewers for their careful evaluation and constructive comments. In response,\nwe have substantially revised the manuscript and performed additional structural, biochemical, and\ncellular experiments to directly address the concerns raised. The major experimental additions and\nanalyses are summarized below:\n(1) We reprocessed the cryo-EM datasets, improving the resolution of the monomeric state from 3.95 Å\nto 3.37 Å and the dimeric state from 3.88 Å to 3.27 Å. These improvements enable more reliable\ninterpretation of intermolecular interactions.\n(2) We performed analytical ultracentrifugation (AUC), providing independent solution-based biochemical\nevidence for the coexistence of monomeric and dimeric assemblies of the CRL2ZYG11B holoenzyme\n(ZYG11B-EloB-EloC-Cul2-Rbx1), consistent with cryo-EM 2D classifications.\n(3) Cellular localization assays demonstrate that both wild-type ZYG11B and the dimer-interface mutant\nlocalize predominantly to the cytoplasm. In parallel, purified CRL2ZYG11B complexes containing either wild-\ntype or mutant ZYG11B co-elute as a single peak by size-exclusion chromatography, indicating intact\nassembly with EloB-EloC-Cul2-Rbx1.\n(4) When expressed at comparable levels with wild-type ZYG11B, disruption of the dimer interface\nsignificantly impairs degradation of degron-fused substrates in cells, as assessed by GPS assays.\n(5) We established a reconstituted in vitro ubiquitination assay using purified proteins. In this system, the\ndimer-interface mutant exhibits markedly reduced CRL2ZYG11B-mediated substrate ubiquitination,\nproviding direct biochemical evidence for the functional relevance of ZYG11B dimerization.\nTogether, these additions substantially strengthen the structural rigor and functional interpretation of the\nstudy and directly address the reviewers’ concerns. Detailed point-by-point responses are provided below\n(reviewers’ comments are shown in red, and our responses in black). Major changes in the revised\nmanuscript are highlighted as underlined text.\n*********************************************************************************************************************\nReviewer #1 (Remarks to the Author):\nThe manuscript by Lin et al. reports very nice cryoEM-derived structures of full-length ZYG11B in complex\nwith other components of this interesting E3 ligase plus a substrate-derived peptide. Prior structures used\nfragments of ZYG11B. While the current manuscript presents compelling structural biology, functional\nassays to support implications of the structural findings are limited or have caveats.\nWe thank the reviewer for the positive assessment of our cryo-EM structures and for highlighting the\nneed for stronger functional validation. In response, we have performed additional in cells and in vitro\nexperiments, including matched-expression GPS assays, biochemical reconstitution of ubiquitination\nactivity, AUC analysis, and complex assembly controls. These data directly strengthen the functional\nimplications of our structural findings and have been incorporated into the revised manuscript.\nBelow are my specific comments, in approximate order of importance.\n1) Figure 4g: I commend the authors for providing the immunoblots to show relative levels of the ZYG11B\nvariants in cells. My concern is that the “dimer surface mutant” was at significantly lower levels than the\nWT protein. What would the data/plot look like if WT levels were matched with the mutant?\nWe thank the reviewer for pointing out this important issue. We agree that unequal expression levels\ncould confound interpretation. To address this concern, we re-established stable cell lines in which wild-\ntype ZYG11B and the dimer-interface mutant are expressed at comparable levels. Under these conditions,\nthe dimer-interface mutant still shows a pronounced defect in promoting degradation of degron-fused\nsubstrates in the GPS assay (below figure and Fig 4h).\n[...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors nicely addressed my comments from the original review. I will only comment further on the cell-free ubiquitination experiments (new data; Supplemental Figure 7). The legend describes the ubiquitination as “robust”. It is not robust so I suggest alternative language. Sometimes these cell-free reactions are weak but if the controls are consistent with E3-dependent ubiquitination (see below), I’d deem these studies acceptable. A couple of notes/suggestions that could enhance cell-free activity. 20 mM ATP is a high concentration—and ATP can bring down the pH of the final reaction substantially if the ATP stock is not pH adjusted or buffered enough. I’d suggest testing 2 mM ATP final and be mindful of the pH. One could also adjust the concentrations of the enzymes and substrate. Also, how were the specific ubiquitin conjugating enzymes chosen? Add a short commentary and cite Li et al. 2024 (PMID: 38382526) or another reference.\nIn the Methods section, the authors write that “reaction mixtures without ATP were used as control”, but I do not see such data. This control, and ideally a control with excess substrate peptide to occupy that pocket on the E3, would strengthen the findings. The \"no ATP\" condition should also show a drastic difference on an anti-ubiquitin blot but the substrate peptide should not affect ubiquitination of non-substrate components (ubiquitination of E3 components is common in these types of reactions) but should reduce your model substrate ubiquitination. An alternative to the excess substrate peptide control would be to leave out the E3. Sometimes 1 or 2 ubiquitins can be added to substrates without an E3 in these types of reactions involving extremely high concentrations of everything. Better overall E3-dependent substrate ubiquitination would allow comparison of multiple concentrations of WT and mutant E3 to estimate how much dimerization impacts E3 activity.\nMinor comment: While “ubiquitination” and “ubiquitylation” are interchangeable terms, it is good practice to use one consistently in a single article.", "reviewer_2": "I am satisfied with the authors’ revisions, which substantially improve the rigor and clarity of the manuscript. The additional structural and biochemical are largely convincing.\nOne more point: While the rebuttal explains why a multi-site mutant was necessary to abolish the dimer interface, this should be briefly stated in the manuscript (better with supplemental data) to clarify why single or limited mutations were insufficient."}, "author_response": "We sincerely thank the reviewers for their constructive suggestions and continued support.\nIn the revised manuscript, we have incorporated additional experimental data and\nclarifications to address the remaining concerns. Detailed point-by-point responses are\nprovided below (reviewers’ comments are shown in red and our responses in black). Major\nrevisions in the manuscript are indicated as underlined text.\nPoint-by-Point Response:\n*************************************************************************************************\nReviewer #1 (Remarks to the Author):\nThe authors nicely addressed my comments from the original review.\nI will only comment further on the cell-free ubiquitination experiments (new data; Supplemental Figure 7). The legend describes the ubiquitination as “robust”. It is not robust so I suggest alternative language. Sometimes these cell-free reactions are weak but if the controls are consistent with E3-dependent ubiquitination (see below), I’d deem these studies acceptable. A couple of notes/suggestions that could enhance cell-free activity. 20 mM ATP is a high concentration—and ATP can bring down the pH of the final reaction substantially if the ATP stock is not pH adjusted or buffered enough. I’d suggest testing 2 mM ATP final and be mindful of the pH. One could also adjust the concentrations of the enzymes and substrate.\nWe thank the reviewer for the continued support and for the valuable suggestions to further\nimprove the biochemical assays. We agree that the ubiquitination activity observed in our\ninitial experiments was modest, as predominantly mono- and di-ubiquitinated substrates\nwere detected without extensive polyubiquitination. Following the reviewer’s\nrecommendation, we optimized reaction conditions, including ATP pH and concentration\nas well as enzyme and substrate levels. Under the revised conditions (5 mM ATP, pH-\nadjusted), substrates remained to be primarily mono- and di-ubiquitinated. However, the\ndimer-interface mutant consistently exhibited markedly reduced activity compared with\nwild-type CRL2ZYG11B (below figure and updated Supplementary Fig 7b). These\noptimizations improve reaction consistency while preserving the key comparative\nconclusion between wild-type and mutant complexes. We have revised the legend of\nSupplementary Figure 7b (updated) to more accurately describe the data as follows: “Wild-\ntype CRL2ZYG11B promoted detectable mono- and di-ubiquitination of the substrate within 5\nmin, whereas the dimer-disrupting mutant showed weaker ubiquitination at all examined\ntime points” (lines 707–710)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I'm satisfied with the revised manuscript. Nice work!"}, "author_response": ""}]

Decision letter