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Structural insights into Ubr1-mediated N-degron polyubiquitination

Abstract

The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1—a single-subunit E3 ligase—is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.

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Fig. 1: The structure of the initiation complex.
Fig. 2: The structure of the elongation complex.
Fig. 3: Analyses of the interactions between Ubc2 and Ubr1.
Fig. 4: A model of Ubr1-mediated polyubiquitination.

Data availability

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB, www.ebi.ac.uk/pdbe/emdb/) under accession codes EMDB-23806 (initiation complex), EMDB-23807 (elongation complex), EMDB-24935 (pre-elongation complex) and EMDB-24936 (apo Ubr1). The atomic models have been deposited in the Protein Data Bank (PDB, www.rcsb.org) under the accession codes 7MEX (initiation complex) and 7MEY (elongation complex). The atomic model of UbcH5 and the RING finger domain of TRIM25 is available under PDB accession code 5FER. Uncropped gels and blots source data are provided in Supplementary Fig. 7. Owing to the large file size, raw electron microscopy data are available from the corresponding authors on request. Source data are provided with this paper.

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Acknowledgements

We thank the staff at the National Cryo-Electron Microscopy Facility at the Frederick National Laboratory and the Advanced Electron Microscopy Facility at the University of Chicago for the help with cryo-EM data collection; H. Rao (Southern University of Science and Technology) and R. Hu (University of Chinese Academy of Sciences) for their help in yeast related experiments. Funding for this work was in part provided by the Catalyst Award C-086 to M.Z. from the Chicago Biomedical Consortium. M.Z. is supported by National Institute of General Medical Sciences of the National Institutes of Health (NIH) under award number R35GM143052. We thank the National Key R&D Program of China (no. 2017YFA0505200) and NSFC (nos 91753205, 81621002, 21621003) for financial support; the National Postdoctoral Program for Innovative Talents (BX2021143), Shuimu Tsinghua Scholar Program (2021SM067) for financial support. This research was in part supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. This research is based on work supported by the National Science Foundation under grant no. 2030381, the graduate research award of Computing and Software Systems division and the start-up fund 74–0525 at University of Washington Bothell to D.S. Any opinions, findings, and conclusions or recommendations expressed in this paper are those of the authors and do not necessarily reflect the views of the National Science Foundation. Molecular graphics and analyses were performed using UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH R01GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

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Authors and Affiliations

Authors

Contributions

M.P., M.Z., L. Liu and Y.Y. designed all of the experiments and interpreted the results. M.P., Q.Z. and L. Liu designed the synthetic route for chemically synthesized ubiquitination initiation and elongation intermediate mimics. T.W. synthesized the fluorescently labelled Ub–Degron and the elongation intermediate mimic. L. Liang synthesized the fluorescently labelled Degron and the initiation intermediate mimic. M.P., Y.Y., D.S. and M.Z. performed cryo-EM data collection and processing. J.M. performed the in vitro ubiquitination assays with Ubr1 and Ubc2 mutants. Q.Z. performed characterization of the U2BR peptide on the enzymatic properties of Ubc2. T.W., Y.Y., C.Z., R.D., J.M., H.A. and Y.X. cloned, expressed and purified Ubr1, Ubc2 and their mutants. M.Z., M.P. and L. Liu wrote the paper. M.Z., L. Liu, Y.Y. and M.P. supervised the project.

Corresponding authors

Correspondence to Man Pan, Yuanyuan Yu, Lei Liu or Minglei Zhao.

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The authors declare no competing interests.

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Peer review information Nature thanks Yong Tae Kwon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Ubr1-mediated Lys48-linked polyubiquitination of degron peptides.

a, The amino acid sequence of the degron peptide (Degron). SPPS: solid-phase peptide synthesis. b, The synthetic route of the monoubiquitinated degron peptide (Ub-Degron). c–d, Fluorescent labelling of Degron (c) and Ub-Degron (d). An additional C-terminal cysteine was introduced for the labelling of fluorescein-5-maleimide. e, In vitro Ubr1-dependent ubiquitination assays using fluorescent Degron (top) and Ub-Degron (bottom) as substrates. Gel images are representative of independent biological replicates (n = 2). f–g, Quantitative evaluations of the kinetics of Ubr1-mediated ubiquitination initiation (f) and the first step of elongation (g). Averages of two independent experiments were plotted and fitted to the Michaelis–Menten model to estimate the Km and Kcat. Gel images are representative of independent biological replicates (n = 2).

Extended Data Fig. 2 Analyses of ubiquitin chain linkage generated by Ubr1 and Ubc2.

a, In vitro Ubr1-dependent ubiquitination on fluorescently labelled K17onlyDegron using wild-type Ub (left) and UbK48R (right). Gel images are representative of independent biological replicates (n = 2). Gel slices in box b and c were cut and digested, followed by LC-MS/MS analyses. b, Identification of Ub chain linkages in box b. c, Identification of Ub chain linkages in box c. # PSMs: Number of peptide spectrum matches.

Source data

Extended Data Fig. 3 Design and purification of the stable intermediate structures.

a, A schematic representation of the transition state of the initiation step. The side chain of a Lys residue on Degron attacks the thioester bond of Ubc2–Ub. The inset shows the designed intermediate structure mimicking the transition state of the initiation step. b, A schematic representation of the transition state of the elongation step. The side chain of Lys48 on Ub-Degron attacks the thioester bond of Ubc2–Ub. The inset shows the designed intermediate structure mimicking the transition state of the elongation step. c, A brief synthetic route of the intermediate structure mimicking the transition state of the elongation step. d, A gel filtration chromatogram of Ubr1 (left) and an SDS–PAGE gel of purified Ubr1 and designed stable intermediate structures Ubc2-Ub-Degron and Ubc2-Ub-Ub-Degron (right).

Extended Data Fig. 4 Cryo-EM density of the initiation and elongation complexes.

a, Individual domains of Ubr1 in the initiation complex. b, Ubc2 and Ub in the initiation complex. c, Ubc2, donor Ub and acceptor Ub in the elongation complex. Maps in a and b were sharpened using a B factor of −96.5 Å2 and contoured at a level of 0.030. Maps in c were sharpened using a B factor of −96.7 Å2 and contoured at a level of 0.022. d, Degron recognition site of the initiation complex. e, Active site of the initiation complex. f, Three-domain junction of the initiation complex. g, Active site of the elongation complex. h, Acceptor Ub and Ubr1 binding interface in the elongation complex. i, Acceptor Ub and Ubc2 binding interface in the elongation complex. Dotted circles in d and e mark the unmodelled densities corresponding to the Degron peptide which are only visible at the lower contour levels. Atomic models could not be reliably built into the densities.

Extended Data Fig. 5 Molecular structures of Ubr1 complex and interfaces between Ubr1 and Ub.

a, The helical scaffold of Ubr1 consists of four separate regions. b, The three domains located around the helical scaffold, Ubr-Box1 (Ubox1, purple), Ubr-Box2 (Ubox2, light blue) and WHD (dark blue). c, The three domains above the helical scaffold, the RING finger domain (cyan), CHD (pink) and UBLC domain (yellow). The RING finger domain is sandwiched between the CHD and UBLC domain. An additional zinc finger motif (ZNF) in UBLC is labelled. d, U2BR (forest green), Ubc2 (magenta)–Ub (lime) and the RING finger domain form the catalytic module of Ubr1 complex. Two zinc finger motifs (ZNF) in the RING finger domain are labelled. e, Additional binding interfaces between the donor Ub and Ubr1 in the initiation complex, including K965 in WHD, E1436 and Q1437 in CHD. R1783 in UBLC domain is the key residue to stabilize the loop of WHD. f, Additional binding interfaces between the acceptor Ub and Ubr1 in the elongation complex. g, In vitro Ubr1-dependent ubiquitination assay. A quadruple mutant (H161A, Y933A, D1175A, and H1763A, DHHY) of the residues involved in the interface between Ubox1, WHD, and UBLC (three-domain junction, shown in Fig. 1e) was tested. Gel images are representative of independent biological replicates (n = 2). h, In vitro Ubr1-dependent ubiquitination assay. Mutants of Ubr1 including K965A, E1436A/Q1437A, R1783A and K965A/E1436A/Q1437A/R1783A (KREQ) involved in the interfaces mentioned in e were tested. Gel images are representative of independent biological replicates (n = 2).

Extended Data Fig. 6 Characterization of N-degron recognition domains on Ubr1.

a, A sequence alignment of Ubr-box1 in yeast and human Ubr1 and human Ubr2. The negatively charged pocket involved in the recognition of the Arg/N-end is highlighted in light purple. b, A close-up view of substrate-engaged Ubr-box1. c, In vitro Ubr1-dependent ubiquitination assay on fluorescently labelled Arg/N-end degron (Degron) and Met/N-end degron (Degron with the first amino acid changed to methionine). Gel images are representative of independent biological replicates (n = 2). d, A structure alignment of yeast Ubr-box2 determined in this study with substrate loaded ClpS (Caulobacter crescentus, 3GQ1) and substrate free ClpS (Plasmodium falciparum, 4O2X). Root-mean-square deviation (RMSD) of backbone atoms are indicated. Substrate peptide from ClpS (Caulobacter crescentus, 3GQ1) is coloured in dark blue. e, A structure alignment of Ubr-box2 determined in this study with substrate loaded ClpS (Caulobacter crescentus, 3GQ1) in the context of Ubr1. The C-terminus of type-1 (this study) and type-2 (from 3GQ1) substrates are highlighted using red and blue arrows, resqectively, pointing to the active site of Ubr1. f, A sequence alignment of yeast Ubr-box2 with ClpS from Caulobacter crescentus (3GQ1) and Plasmodium falciparum (4O2X). The substrate binding pocket is highlighted in yellow.

Extended Data Fig. 7 Characterization of the interfaces between Ubr1, Ubc2 and Ub.

a–b, In vitro Ubr1-dependent ubiquitination assays. Mutations of the Ub binding loop on Ubr1 (H678A/V679A/L680A/H681A, named UBLM mutant, a) and Ubc2 (N123A/V124A, b) were tested. Gel images are representative of independent biological replicates (n = 2). Red boxes highlight the difference of UBLM mutant in initiation and elongation. c, Single-turnover ubiquitination assay of wild-type Ubr1 and UBLM mutant using Ubc2 charged with either wild-type Ub or UbK0 (all Lysine residues mutated to Arginine). Red boxes highlight the defect of UBLM mutant in elongation. Gel images are representative of independent biological replicates (n = 2). d, Side views of the initiation and elongation complexes showing the displacement of U2BR, Ubc2, and Ub. e, An alignment of Ubr1 structures in the initiation and elongation complexes. f–g, In vitro Ubr1-dependent ubiquitination assays. Gel images are representative of independent biological replicates (n = 2). f, Ubr1 (F1190A/Q1186A/F1183A/H1175A, named FQFH mutant) and Ubc2 mutants at the interface shown in Fig. 3b were tested. g, The inhibition of Ubr1-dependent ubiquitination in the presence of increasing concentrations of a synthetic U2BR peptide. h, The accessibility of the catalytic cysteine (Cys88) of Ubc2 was tested using fluorescein-5-maleimide, a bulky fluorescent alkylation reagent (BFAR), in the presence or absence of the synthetic U2BR peptide. The average fluorescence from two independent biological replicates was plotted (n = 2). i, A sequence alignment of multiple E2 enzymes, including yeast and human Ubc2 (also known as Rad6b in human). Two regions involved in the interaction with the RING finger domain are shown. j, In vitro Ubr1-dependent ubiquitination assays were performed to examine the role of Asn65 of Ubc2 in the interaction with the RING finger domain of Ubr1. Gel images are representative of independent biological replicates (n = 2).

Extended Data Fig. 8 Characterization of the interactions between U2BR and Ubc2.

a, ITC measurement of the binding between Ubc2 and the synthetic U2BR peptide. b, The formation of E1-dependent Ubc2–Ub thioester in the presence of the synthetic U2BR peptide. Gel images are representative of independent biological replicates (n = 2). c, Quantitative evaluations of the inhibitory effect of the synthetic U2BR peptide on E1-dependent Ubc2–Ub thioester formation. Averages of three independent biological replicates (n = 3) were plotted and fit to estimate the IC50 of the synthetic U2BR peptide. The curves are presented as mean values ± s.d. d–e, In vitro Ubr1-dependent ubiquitination assay. Increasing doses of catalytically inactive Ubc2-C88S (d) and non-hydrolysable Ubc2-Ub (e) up to 12.5 μM were pre-mixed with Ubr1, followed by adding wild-type Ubc2 at 4 μM. Gel images are representative of independent biological replicates (n = 2).

Extended Data Fig. 9 The structure of the pre-elongation complex and validation of the structural mechanism.

a, A comparison of initiation, pre-elongation and elongation complex in the same orientation showing the movement of ubiquitin. The colour code of Ubr1 is the same as that in Fig. 1b. Sharpened map of the pre-elongation complex is shown at a contour level of 0.011. b, In vitro ubiquitination assay on fluorescently labelled Hs-Type-1 Degron (derived from human protein Rec8) with wild-type Ubr1 and Ubr1 mutants, UBLM, DHHY and FQFH. Gel images are representative of independent biological replicates (n = 2). c, In vitro ubiquitination assay on fluorescently labelled Type-2 Degron (derived from Sindbis virus polymerase nsP4) with wild-type Ubr1 and Ubr1 mutants, UBLM, DHHY and FQFH. Gel images are representative of independent biological replicates (n = 2). d, In vitro ubiquitination assay on truncated protein substrates ROQ1 (22-104) and Scc1 (268-384) with wild-type Ubr1 and Ubr1 mutants, UBLM, DHHY and FQFH. Gel images are representative of independent biological replicates (n = 2). e, The design of the yeast-growth assay. Endogenous deubiquitinating enzymes (DUB) cleave the construct co-translationally and produce Scc1 fragment (R-Scc1269–566) which has an N-terminal arginine residue and is toxic to the yeast strain deficient of Ubr1 (ΔUBR1). If wild-type Ubr1 is supplemented, R-Scc1269–566 will be rapidly polyubiquitinated and degraded, reversing the growth defect. f. Yeast strains carrying wild-type Ubr1 or Ubr1 mutants (FQFH, UBLM and DHHY) were streaked either on dextrose-containing (SD) plates (right) where all strains grew without the expression of Scc1 fragment and Ubr1 variants, or on galactose containing (SG) plates (left), where all strains grew with the induced gene expression of Scc1 fragment and Ubr1 variants. In addition to R-Scc1269–566, M-Scc1269–566 which has an N-terminal methionine residue was also tested. The plates were incubated at 30 °C for 3 d.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Pan, M., Zheng, Q., Wang, T. et al. Structural insights into Ubr1-mediated N-degron polyubiquitination. Nature 600, 334–338 (2021). https://doi.org/10.1038/s41586-021-04097-8

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