Structural visualization of HECT-type E3 ligase Ufd4 accepting and transferring ubiquitin to form K29/K48-branched polyubiquitination

Abstract

The K29/K48-linked ubiquitination generated by the cooperative catalysis of E3 ligase Ufd4 and Ubr1 is an enhanced protein degradation signal, in which Ufd4 is responsible for introducing K29-linked ubiquitination to K48-linked ubiquitin chains to augment polyubiquitination. How HECT-E3 ligase Ufd4 mediates the ubiquitination event remains unclear. Here, we biochemically determine that Ufd4 preferentially catalyses K29-linked ubiquitination on K48-linked ubiquitin chains to generate K29/K48-branched ubiquitin chains and capture structural snapshots of Ub transfer cascades for Ufd4-mediated ubiquitination. The N-terminal ARM region and HECT domain C-lobe of Ufd4 are identified and characterized as key structural elements that together recruit K48-linked diUb and orient Lys29 of its proximal Ub to the active cysteine of Ufd4 for K29-linked branched ubiquitination. These structures not only provide mechanistic insights into the architecture of the Ufd4 complex but also provide structural visualization of branched ubiquitin chain formation by a HECT-type E3 ligase.

Fig. 1. Ufd4 prefers to synthesize K29/K48-branched ubiquitin chains on K48-linked ubiquitin chains.

a Ufd4-dependent in vitro ubiquitination on fluorescent MonoUb and K48-linked diUb. Gel images and columns of relative conversions (%) are shown. Gel images are representative of independent biological replicates (n = 2). b Schematic representation of the identification flow of Ub topology generated by Ufd4 on K48-linked Ub chains. The blue symbols represent the isopeptide bond between Lys48 and Ub C-terminal, the red symbols represent the isopeptide bond between Lys29 and Ub C-terminal, the grey symbols represent Ub C-terminals. c Intact MS analysis of in vitro ubiquitination reactions treated with Lb^pro*, and the spectra were deconvoluted. Deconvoluted spectra (left), quantification of the relative abundance of each ubiquitin species (right) are shown. Data represent the mean ± SD of three independent experiments. d Identification of K29/K48-branched ubiquitin chains. The MS/MS spectrum corresponding to the signature peptide derived from K29/K48-branched linkages (aa 29–57, GlyGly modified at K29 and K48) was obtained. e Ufd4-dependent in vitro ubiquitination on fluorescent K48-linked diUb with lysine to arginine mutation at the proximal or distal Lys29 sites. Gel images are representative of independent biological replicates (n = 3). f The Ufd4’s ubiquitination activity on K48-linked triUb with a K29-only residue in the proximal, middle or distal Ub. Gel images are representative of independent biological replicates (n = 2). Source data are provided as a Source Data file.

Fig. 2. Overall structure of Ufd4 in complex with triUb^K29/K48.

a Schematic of two-step complex assembly routes of Ufd4 in complex with triUb^K29/K48. Step one: synthetic route of the intermediate structure mimicking the transition state of Ufd4-mediated ubiquitination at proximal Lys29 site of K48-linked diUb. Step two: in vitro crosslinking experiments between Ufd4 and triUb^K29/K48. b Schematic diagram of the native transition state vs. the transition state mimic of the ubiquitination process. The side chain of Lys29 residue on K48-linked diUb attacks the thioester bond of Ufd4–Ub. An intermediate structure design is shown in the inset to mimic the transition state of Ufd4-mediated Ub chain assembly. c Structural domain diagram of Ufd4 with indicated residue boundaries. The dashed-line box indicates the unresolved N-terminus region, the turquoise box represents the Ub binding domains, the dusty blue box indicates the HECT domain, and the red line represents the active Cys1450. d, e Cryo-EM density (d) and structural model (e) of the Ufd4-triUb^K29/K48 complex.

Fig. 3. Analysis of the interactions between Ufd4 and triUb^K29/K48.

a Cryo-EM density of the ARM region and the HECT C-lobe with K48-linked diUb, dashed regions correspond to interfaces between Ufd4 and Ub^K48−dist, Ufd4 and Ub^K48−prox. b–d Molecular interactions between the ARM region and Ub^K48−dist (b), the ARM region and Ub^K48−prox (c), the HECT C-lobe and Ub^K48−prox (d). e Cryo-EM density of the HECT C-lobe with donor Ub. f Molecular interactions between the HECT C-lobe and donor Ub. g, h Cryo-EM density of the HECT C-lobe and triUb^K29/K48 (g), and the structural model showing the spatial proximity of HECT C-lobe Cys1450, Lys29 of Ub^K48–prox and C-terminal of donor Ub (h). i, j In vitro Ufd4-dependent ubiquitination assays. Mutants at the substrate recognition interface (i) and the donor Ub interface (j) were tested. Gel images are representative of independent biological replicates (n = 3). Source data is provided as a Source Data file.

Fig. 4. Analysis of the interactions between TRIP12 and triUb^K29/K48.

a Cryo-EM density (a) and structural model (b) of the TRIP12-triUb^K29/K48 complex. c Structural alignment of Ufd4-triUb^K29/K48 complex and TRIP12-triUb^K29/K48 complex structures, and only ARM region, C-lobe and K48-linked diUb are shown. d, e Molecular interactions between the ARM region and Ub^K48−dist (d), the ARM region and Ub^K48−prox (e). f–h Molecular interactions between the C-lobe and Ub^K48−prox.

Fig. 5. The structure of Ufd4 in complex with Ubc4-Ub.

a Schematic of two-step complex assembly routes of Ufd4 in complex with Ubc4-Ub. Step one: synthetic route of the intermediate structure mimicking the transition state of Ufd4-mediated transthiolation from Ubc4-Ub. Step two: in vitro crosslinking experiments between Ufd4 and Ubc4-Ub. b Schematic diagram of the native transition state vs. the transition state mimic of the transthiolation process. The side chain of catalytic Cys residue on Ufd4 attacks the thioester bond of Ubc4–Ub. An intermediate structure design is shown in the inset to mimic the transition state of the Ufd4-mediated transthiolation process. c Cryo-EM density and structural model of the Ufd4^ΔARM-Ubc4 complex. d–g Molecular interactions between the HECT C-lobe and Ubc4 (d), the HECT N-lobe and Ubc4 (e–g). i, j In vitro Ufd4~Ub thioester formation assays. Ubc4 mutants (i) and Ufd4 mutants (j) were tested. Gel images are representative of independent biological replicates (n = 2). Source data are provided as a Source Data file.