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Structural basis for E4 enzyme Ufd2-catalyzed K48/K29 branched ubiquitin chains

Nature Chemical Biology volume 22, pages 239–248 (2026)Cite this article

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Abstract

E4 enzymes amplify and remodel ubiquitin chain signals beyond the conventional E1–E2–E3 cascade. The first identified E4 enzyme Ufd2 preferentially catalyzes K48/K29 branched ubiquitin chains, yet the structural mechanism remains unknown. Here, we combined chemical biology and cryo-electron microscopy to visualize stable intermediates in Ufd2 loading ubiquitin at K48 of proximal ubiquitin on K29-linked di- and triubiquitin. Our data reveal that the core region of Ufd2 functions as an unprecedented K29 diubiquitin binding domain, interacting extensively with proximal and distal ubiquitin, which orients the K48 site of proximal ubiquitin toward the active site of Ubc4, facilitating K48/K29 branched ubiquitin chain formation. We also identified a unique dimeric conformation where dimerized Ufd2 and Ubc4 stabilize each other’s distal ubiquitin during branching on K29 triubiquitin. Our findings provide mechanistic insights into the assembly of K48/K29 branched ubiquitin chains by the E4 enzyme Ufd2 and highlight the spatial cooperation among multiple pairs of ubiquitin-related enzymes on longer ubiquitin chains.

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Fig. 1: Capturing the complex of Ufd2-mediated K29diUb branched ubiquitination.
Fig. 2: Analysis and validation of the interactions between Ufd2 and K29diUb.
Fig. 3: Analysis and validation of the interactions between Ufd2 and Ubc4.
Fig. 4: Structural and biochemical analysis of the triUb dimeric complex.
Fig. 5: Cartoon model of Ufd2-mediated K48/K29 branched ubiquitination.

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Data availability

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (www.ebi.ac.uk/pdbe/emdb/) under accession codes EMD-62353 (diUb monomeric complex), EMD-62354 (triUb dimeric complex), EMD-62355 (triUb monomeric complex) and EMD-62356 (diUb dimeric complex). The atomic models have been deposited in the PDB (www.rcsb.org) under accession codes 9KHS (diUb monomeric complex), 9KHT (triUb dimeric complex) and 9M7O (triUb monomeric complex). Source data are provided with this paper. The source data for the MS analysis in Extended Data Fig. 2f–h have been uploaded to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD062799) under accession number PXD062799.

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Acknowledgements

We thank the National Natural Science Foundation of China (92253302, T2488301, 22137005 and 22227810 to L.L.; 22277073 and 92253302 to M.P.), National Key R&D Program of China (2022YFC3401500 to L.L.; 2023YFA0915300 to M.P.), New Cornerstone Science Foundation (to L.L.), Shanghai Rising-Star Program (22QA1404900 to M.P.), Shanghai Pilot Program for Basic Research–Shanghai Jiao Tong University (21TQ1400224 to M.P.), Foundation of Muyuan Laboratory (118602240 to M.P.), Fundamental Research Funds for the Central University (to M.P.), Shanghai Jiao Tong University 2030 Initiative (WH510363003/003 to M.P.), the Shanghai Natural Science Foundation (25ZR1402193) and Shanghai Frontiers Science Center of Drug Target Identification and Delivery (ZXWH2170101 to H.A.). We acknowledge the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for cryo-EM screening and data collection from 200-kV Arctica Tecnai and 300-kV Titan Krios microscopes. We thank the laboratory of W. Li (Guangzhou Medical University) for supporting our yeast experiments.

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Author notes

  1. Zebin Tong

    Present address: Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany

  2. These authors contributed equally: Zebin Tong, Xiangwei Wu, Hongyi Cai.

Authors and Affiliations

  1. Institute of Translational Medicine, School of Pharmaceutical Sciences, School of Chemistry and Chemical Engineering, National Center for Translational Medicine (Shanghai), Shanghai Key Laboratory for Antibody-Drug Conjugates with Innovative Target, Shanghai Jiao Tong University, Shanghai, China

    Zebin Tong, Xiangwei Wu, Hongyi Cai, Zhiheng Deng, Huasong Ai & Man Pan

  2. New Cornerstone Science Laboratory, Tsinghua-Peking Joint Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Center for Synthetic and Systems Biology, Department of Chemistry, Tsinghua University, Beijing, China

    Zebin Tong, Xiangwei Wu, Hongyi Cai, Shidian Wu, Tianyi Zhang, Zhiheng Deng, Ziyu Xu, Rujing Yuan & Lei Liu

  3. Center for Future Foods, Muyuan Laboratory, Zhengzhou, China

    Man Pan

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Contributions

Z.T., X.W., H.A., L.L. and M.P. proposed the idea, designed the experiments and analyzed the results. X.W., H.C., T.Z. and Z.T. cloned the plasmids. Z.T., X.W., H.C. and S.W. expressed the proteins (Ufd2 and mutants, Ubc4 and mutants and Ub variants). X.W. and H.C. synthesized the K29diUb–Ubc4–Ub and K29triUb–Ubc4–Ub probes. Z.T., H.C. and X.W. prepared the cryo-EM samples and collected the cryo-EM data. H.A. and Z.T. processed the cryo-EM data and determined the cryo-EM structures of the Ufd2–K29diUb–Ubc4–Ub and Ufd2–K29triUb–Ubc4–Ub complexes. Z.T. built the atomic models. H.C. and X.W. synthesized fluorescently labeled K29diUb and their variants and fluorescently labeled K29triUb. Z.T., X.W. and H.C. performed the in vitro ubiquitination assay. X.W. and H.C. performed SPR experiments, MS analyses and expression shutoff analyses. Z.T. performed the SEC–MALS and BS3 analyses. Z.T., X.W. and H.C. collated the experimental data and prepared the figure panels and tables. Z.T. drafted the paper. Z.T., X.W., H.C., H.A., L.L. and M.P. revised the paper. Z.T., X.W., H.C., S.W., T.Z., Z.D., Z.X., R.Y., H.A., L.L. and M.P. read, discussed and analyzed the paper. H.A., L.L. and M.P. supervised the project.

Corresponding authors

Correspondence to Huasong Ai, Lei Liu or Man Pan.

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Nature Chemical Biology thanks Helen Walden and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The possible model of Ufd2/Ubc4-Ub/K29diUb complex generated based on previous biochemical and structural results.

a, Crystal structure (PDB: 3L1Z) of the U-box domain of human UBE4B Ub ligase (Ufd2 homolog) in complex with the E2 enzyme UBCH5C (Ubc4 homolog, PDB:1QCQ). b, Crystal structure of apo Ufd2 (PDB: 2QJ0). c, Crystal structure of K29diUb (PDB:4S22). d, Crystal structure of Ufd2 complex with ubiquitin-like (UBL) domain of Rad23 (PDB:3M62). e, Superimposition of the structure in (a) and (b), aligned with U-box domain; Ubc4 (PDB: 1QCQ) was structural aligned with UBCH5C. f, Superimposition of the structure in (c) and (d), aligned the distal Ub with UBL domian. g, Superimposition of the structure in (e) and (f), aligned with Ufd2. The distance between the catalytic cysteine of Ubc4 and the distance between the catalytic cysteine of Ubc4 and the side-chain amine of the acceptor Ub K48 residue is 103 Å.

Extended Data Fig. 2 UFD2 prefers to synthesize the K48/K29 branched Ub chain.

a, In vitro Ufd2-dependent ubiquitination assays on K29diUb and K29triUb were performed (E1: Uba1, E2: Ubc4). b, In vitro Ufd2-dependent ubiquitination assays were performed on fluorescent K29diUb mutants. K29diUbPro* refers to K29diUb with lysine to arginine mutation at the proximal Lys48 site and labeled with Oregon Green 488 dye (OG488). K29diUbDis* refers to K29diUb with lysine to arginine mutation at the distal Lys48 site and labeled with OG488. K29diUbWT refers to K29diUb without any mutation at Lys48 site and labeled with OG488. The synthetic routes were depicted in Supplementary Fig 8. c, In vitro Ufd2-dependent ubiquitination assays on eight different linkage-type diUbs were performed. d, The purified products of in vitro Ufd2-dependent ubiquitination reactions on K29diUb and K29triUb were treated with the protease LbPro*. The above gels (a-d) are representative of three independent experiments. e, A schematic representation illustrates the mass spectrometry identification workflow for the Ub chain architectures generated by Ufd2 on K29diUb and K29triUb. f, The representative MS/MS spectrum corresponding to the signature peptide derived from K29-K48 branched linkages (aa 29–57), featuring GlyGly modifications at K29 and K48, is presented. g-h, Intact mass spectrometry (MS) analysis of Lbpro* treated in vitro ubiquitination reactions, and quantification of the relative abundance of each ubiquitin species was shown in (g) using K29diUb as substrate and (h) using K29triUb as substrate from n = 2 independent experiments. Each data point is shown as a black dot [data from (g)].

Extended Data Fig. 3 Synthetic route of K29triUb-Ubc4-Ub probe and characterization of chemical probes.

a, A schematic representation illustrates the native state of the ubiquitination process catalyzed by Ufd2 using K29triUb as substrate. The side chain of the Lys48 residue on the proximal Ub of K29triUb attacks the thioester bond of Ubc4~Ub. b, The designed chemical mimic of ubiquitylation intermediate, termed K29triUb-Ubc4-Ub probe, and a schematic outline of the three-step complex assembly routes for the K29triUb-Ubc4-Ub probe is presented. Step one: CAET-assisted synthesis of the intermediate mimicking Ubc4~Ub thioester, termed Ubc4-Ub-SH. Step two: Activation of the proximal Cys48 residue in the K29triUbprobe to yield the intermediate, termed K29triUbprobe-TNB. Step three: disulfide bond coupling of above two intermediates generates the K29triUb-Ubc4-Ub probe. c, Structural formula of DTNB and CAET-Acm which used for synthesis of chemical probes. d-e, A representative gel filtration chromatography of disulfide bond coupling products separated using Superdex 75 Increase 10/300 GL (λ = 260 and 280 nm), with the product peak marked by a yellow block in (d) for K29diUb-Ubc4-Ub and in (e) for K29triUb-Ubc4-Ub and its SDS–PAGE characterisation. This gel are representative of two independent experiments. f-g, Deconvoluted mass spectra of K29diUb-Ubc4-Ub in (f) and K29triUb-Ubc4-Ub in (g) are presented. The symbol * represents peak of the target product.

Extended Data Fig. 4 Single-particle cryo-EM analysis of Ufd2/K29diUb-Ubc4-Ub complex.

a, A representative cryo-EM micrograph from the dataset in Ufd2/K29diUb-Ubc4-Ub complex from a dataset of 6640 images. b, CTF estimation of the micrograph depicted in (a). c, Representative 2D classification of the Ufd2/K29diUb-Ubc4-Ub complex. d, Left: representative cryo-EM images of the Ufd2/K29diUb-Ubc4-Ub complex, featuring particles identified through auto-picking, highlighted by green circles. Right: cryo-EM data processing of Ufd2/K29diUb-Ubc4-Ub complex. The dataset underwent particle selection, 2D classification, and multiple rounds of 3D classification. The workflows are depicted with a red background representing the computational process of the monomer conformation, whereas a blue background illustrates the computational process of the dimer conformation.

Extended Data Fig. 5 Cryo-EM density of the Ufd2/K29diUb-Ubc4-Ub complex.

a-b, The local resolution of the map calculated using Relion and distribution of the Euler angles within diUb monomeric complex (a) and diUb dimeric complex (b). c, In cryo-EM, the Fourier shell correlation (FSC) curve is used to assess the resolution of the reconstructed 3D density map. By comparing two different half-maps, the FSC evaluates their similarity to determine the resolution, with an FSC = 0.143 serving as a standard threshold for ascertaining the final average resolution. The Y-axis represents correlation, while the X-axis is the inverse of resolution. The FSC = 0.143 show that the overall resolution of 3.99 Å and 4.31 Å for diUb monomeric complex, with a resolution of 7.84 Å for diUb dimeric complex after RELION postprocessing. d, The FSC curve is employed to evaluate the similarity between the model and the cryo-EM map, with an FSC value of 0.5 indicating that the model is reasonable at this specific resolution. The FSC curve demonstrates that the resolution of the diUb monomeric complex at FSC = 0.5 is 4.31 Å. e, Representative regions of cryo-EM densities for Ufd2 core region (188-879), Ufd2 U-box (880-961), Ubc4 (1-148), UbDon (1-76), UbPro (1-76), UbDis (1-76) Ubc4-UbDon-UbPro and K29diUb-UBH interface.

Extended Data Fig. 6 Structural analysis of diUb dimeric complex.

a-b, Structure model of diUb dimeric complex with semi-transparent cryo-EM density maps and rectangular regions correspond to the interfaces shown in (e). c, Interface between core regionA and core regionB shown in (f). d, Representative two-dimensional (2D) characteristic of the diUb dimeric complex. e, Expanded view of the head-to-head assembled dimeric Ufd2. f, Expanded view of the formation of dimeric Ufd2 mediated by the dimeric helix (residue 419-439). g, Cryo-EM density of diUb dimeric complex at level 0.02, the density is represented in a translucent manner and fitted in two diUb monomeric complex (PDB:9KHS) and two N-terminal variable domain of Ufd2 (PDB: 3M62). h, A side view of the cryo-EM density of the di-ubiquitin dimeric complex. i. A close-up view of the N-terminal region of the di-ubiquitin dimeric complex reveals that the N-terminal variable domains of Ufd2 in the two protomers do not conflict.

Extended Data Fig. 7 Analysis of the interactions between Ufd2 and K29diUb.

a, Superimposition of the crystal structure of K29diUb (PDB: 4S22) and K29diUb determined in this study, with alignment of the proximal Ub molecules in both structures. b, K29diUb determined in the diUb monomeric complex, as well as its complexes with sAB-K29 (PDB: 7EKO) and NZF1 (PDB: 4S1Z), with the distance between the C-terminus of UbPro and the N-terminus of UbDis. c, Distribution of classical interaction patches on K29diUb that engage in interactions within the Ufd2/K29diUb complex is illustrated. I44 patch is depicted in dark blue, I36 patch in dark green, F4 patch in magenta and TEK-box in orange. d, Distribution of classical interaction patches on K29diUb that engage in interactions within the sAB-K29/ K29diUb complex is illustrated. The colour code of the hydrophobic patches is the same as that in (c). e, Distribution of classical interaction patches patches on K29diUb that engage in interactions within the sAB- NZF1/K29diUb complex is illustrated. The colour code of the hydrophobic patches is the same as that in (c). f, A table listed comparison of the classical interaction patches patches on K29diUb that engage in interactions with Ufd2, sAB-K29, and NZF1, highlighting the differences and similarities in their engagement patterns. g, The SPA motifs of different E2 enzymes are conserved in their interactions with the RING/U-box domains. h, A sequence alignment of representative RING domains and U-box domains reveals the conserved linchpin residue, which is highlighted by red solid circle.

Extended Data Fig. 8 Single-particle cryo-EM analysis of Ufd2/K29triUb-Ubc4-Ub complex.

a, A representative cryo-EM micrograph from the dataset in Ufd2/K29triUb-Ubc4-Ub complex from a dataset of 7112 images. b, CTF estimation of the micrograph depicted in (a). c, Representative 2D classification of the Ufd2/K29triUb-Ubc4-Ub complex. d, Left: representative cryo-EM images of the Ufd2/K29triUb-Ubc4-Ub complex, featuring particles identified through auto-picking, highlighted by green circles. Right: cryo-EM data processing of Ufd2/K29triUb-Ubc4-Ub complex. The dataset underwent particle selection, 2D classification, and multiple rounds of 3D classification. The workflows are depicted with a red background representing the computational process of the monomer conformation, whereas a blue background illustrates the computational process of the dimer conformation. e-f, The distribution of the Euler angles within triUb monomeric complex (e) and triUb dimeric complex (f). g, The Fourier Shell Correlation (FSC) plots derived from half-maps, along with the mask employed for the determination of the average resolution at an FSC threshold of 0.143, showing the overall resolution of 4.14 Å for triUb monomeric complex and 4.85 Å for triUb dimeric complex.

Extended Data Fig. 9 Validation of Ufd2 interactions with K29triUb and exploration of Ufd2 dimerization.

a, Structure model of triUb monomeric complex. b, In vitro Ufd2-dependent ubiquitination assays using fluorescent labeled K29triUb as substrate. Various Ufd2 mutations, including L347G, F361G, Y204R, D365K, D369K, D299K, H300G, D299K&H300G, H576G, and D360A, were analyzed. The gel image is representative of independent biological replicates (n = 3). c, Bar graph comparing the fraction of reacted K29triUb and show the mean ± SD from n = 3 independent experiments. Each data point is shown as a black dot [data from (b)]. A two-sample, two-tailed Student’s unpaired t test was employed to calculate p values; ****p < 0.0001. d, A representative size-exclusion chromatography with multi-angle light scattering (SEC-MALS) of Ufd2, with peaks corresponding to the Ufd2 dimer and monomer labeled. e, Surface Plasmon Resonance (SPR) measurement of binding of wild-type Ufd2 to wild-type Ufd2 or Ufd2 dimer mutant lacking the dimeric helix (residue 419-439). f, Bis(sulfosuccinimidyl) suberate (BS3) crosslinking analysis of wild-type Ufd2 and Ufd2 dimer mutant. These gels are representative of two independent experiments.g, In vitro Ufd2-dependent ubiquitination assays were performed to compare the ubiquitination efficiency of Ubc4 wild-type and Ubc4 S23R, utilizing fluorescently labeled K29-triUb as the substrate. Gel images are representative of independent biological replicates (n = 3). h, The line graph illustrates the proportions of K29-triUb in the reactions, and show the mean ± SD from n = 3 independent experiments.

Extended Data Fig. 10 Cryo-EM density of the triUb dimeric complex.

a, Cryo-EM density of Ufd2B-Ufd2A scaffold in the triUb dimeric complex, with the density of dimer helix highlighted in a box. b, Colored cryo-EM density of K29triUb, consisting of UbPro, UbMid, and UbDis, in the triUb dimeric complex. c, Cryo-EM density of catalytic assembly consisting of Ubc4, UbDon, UbPro and U-box in triUb dimeric complex. d, Cryo-EM density of backside UbDis-B -Ubc4A interface in the triUb dimeric complex. e, Cryo-EM densities for Ufd2A (1-961) and Ufd2B (1-961) in the triUb dimeric complex. f, Representative regions of cryo-EM densities for Ufd2 U-boxA (880-961), Ufd2 U-boxB (880-961), Ubc4A (1-148), Ubc4B (1-148), UbPro-A (1-76), UbPro-B (1-76), UbMid-A (1-76), UbMid-B (1-76), UbDis-A (1-76), UbDis-B (1-76), UbDon-A (1-76) and UbDon-B (1-76).

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Supplementary Figs. 1–12, Tables 1–7 and uncropped scans of blots and gels in the Supplementary Information.

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Uncropped gels.

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Uncropped gels.

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Tong, Z., Wu, X., Cai, H. et al. Structural basis for E4 enzyme Ufd2-catalyzed K48/K29 branched ubiquitin chains. Nat Chem Biol 22, 239–248 (2026). https://doi.org/10.1038/s41589-025-01985-2

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