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Atomic structure of the APC/C and its mechanism of protein ubiquitination

Abstract

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C–coactivator complexes with either Emi1 or a UbcH10–ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10–ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo.

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Figure 1: EM reconstructions of the APC/CCdh1.Emi1 complex.
Figure 2: The C box of Cdh1 and IR tails of Cdh1 and Apc10 interact with structurally related sites on Apc8 and Apc3.
Figure 3: Interactions of Emi1 with Apc2CTD–Apc11RING and D-box receptor of Cdh1 and Apc10.
Figure 4: Structure of the APC/CCdh1.Hsl1.UbcH10–Ub complex reveals the location of UbcH10.
Figure 5: The relative position of the catalytic and substrate-recognition modules defines the target lysine.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

EM maps have been deposited in the Electron Microscopy Data Bank under accession codes 2924 (APC/CCdh1.Emi1), 2925 (APC/CCdh1.Hsl1.UbcH10–Ub) and 2926 (APC/CCdh1.Hsl1.Apc11–UbcH10). APC/CCdh1.Emi1 coordinates have been deposited in the Protein Data Bank under accession number 4UI9.

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Acknowledgements

This work was funded by a Cancer Research UK grant to D.B. We thank W. J. Chazin and members of the Barford group for discussions, and X. Bai and S. Scheres for their help with RELION; C. Savva and S. Chen for EM facilities; P. Emsley for help with COOT; G. Murshudov for help with REFMAC; G. McMullan for assistance in movie data capture; J. Grimmett and T. Darling for computing; and A. Boland for advice with COOT and PyMol.

Author information

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Authors

Contributions

L.C. prepared grids, collected and analysed EM data and determined the three-dimensional reconstructions, fitted coordinates and built models, prepared figures and co-wrote the paper. Z.Z. designed and made constructs, performed biochemical analysis and purified proteins. J.Y. prepared and purified the complexes and performed biochemical analysis. S.H.McL. performed and analysed surface plasmon resonance experiments. D.B. directed the project, built models and co-wrote the paper.

Corresponding author

Correspondence to David Barford.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Preparations and EM images of APC/C complexes.

a, Coomassie-blue-stained SDS gel of APC/CCdh1.Emi1. b, Coomassie-blue-stained SDS gel of APC/CCdh1.Hsl1.Apc11–UbcH10. c, Coomassie-blue-stained SDS gel and western blot analysis (anti-His antibody—only ubiquitin in the complex contains His tag) of APC/CCdh1.Hsl1.UbcH10–Ub without or with cross-linking by glutaraldehyde. d, A typical cryo-EM micrograph of APC/CCdh1.Emi1; representative of 3,328 micrographs. e, Gallery of two-dimensional averages of APC/CCdh1.Emi1 showing different views; representative of 100 two-dimensional averages. f, Local resolution map of APC/CCdh1.Emi1 showing resolution range. g, Details of EM density for segments of α-helix and β-strand of Apc1 and the C box of Cdh1.

Extended Data Figure 2 Resolution estimation and example of de novo model building.

a, Gold-standard FSC curve and FSC curve between cryo-EM map and final atomic model of the APC/CCdh1.Emi1. b, Cross-validation of model refinement by half maps. Shown are FSC curves between the atomic model and the half map (maphalf1) it was refined against, and FSC curves between the atomic model and the other half map (maphalf2) that was not used during refinement. c, Gold standard FSC curve of APC/CCdh1.Hsl1.UbcH10–Ub. d, Gold-standard FSC curves of human APC/CCdh1.Hsl1.Apc11–UbcH10. e, De novo model building of Apc15 N-terminal loop. The surrounding Apc5 residues are also shown. f, EM density for the LR tail common to Emi1 and Ube2S is only observed in APC/C complexes with subunits incorporating the LR tail. (1) APC/CCdh1.Emi1, (2) An APC/C complex with an LR tail-bearing subunit (UbcH10LR of APC/CCdh1.Hsl1.UbcH10–Ub), (3) No LR tail density in APC/CCdh1.Hsl1.Apc11–UbcH10 fusion and (4) APC/CCdh1.Hsl1 (ref. 4).

Extended Data Figure 3 Apc1 structure and the TPR lobe interacts with multiple subunits.

a, Cartoon of Apc1 colour-ramped from blue to red for the N to C termini. Insertions that interact with Apc2, Apc8 and Apc10 are labelled. Apc1Mid adopts a novel architecture. b, TPR lobe with TPR subunits shown as surface representations, with the small TPR-accessory subunits (Apc12, Apc13, Apc15 and Apc16), a segment of Apc5, IR tails of Cdh1 and Apc10, and the Cdh1 C box that interact with the TPR lobe are shown as cartoons. The N termini of Apc12, Apc13 and Apc15 are buried. The eight structurally homologous and symmetry-equivalent sites on the TPR lobe that bind Apc13, Apc16 and Apc5 are indicated and shown in detail in c. The view is similar to Fig. 1a. c, The eight TPR subunits interact with Apc13, Apc16 and Apc5 mainly through contacts to four conserved aromatic residues present on most TPR subunits (Y308, Y309, W302, Y322 of Apc6 (panels 5 and 6)). d, Sequence of the ordered region of Cdh1NTD bound to Apc1 and Apc8 (ordered regions shown as lines and α-helices). Critical Apc1 and Apc8 contact residues are indicated with green and blue arrows. Phosphorylation sites are indicated with red arrows.

Extended Data Figure 4 APC/C ubiquitination assays.

a, Mutation of Arg493 of the IR tail reduces APC/CCdh1 activity. b, Mutations at the RING domain interface of UbcH10 and UbcH10LR disrupt ubiquitination activity. c, Ubiquitination assay shows that both UbcH10(C114K) and UbcH10LR(C114K) compete with wild-type UbcH10. UbcH10LR(C114K) is a more potent inhibitor. d, The APC/C–UbcH10-mediated substrate ubiquitination activities of UbcH10 and UbcH10LR are indistinguishable. e, The ubiquitin (I36A) and ubiquitin (I44A) mutants were defective for APC/C–UbcH10-mediated substrate ubiquitination. Experiments in Extended Data Fig. 4a–e were replicated three times. f, UbcH10 charging by the ubiquitin (I36A) and ubiquitin (I44A) mutants was unchanged relative to wild-type ubiquitin.

Extended Data Figure 5 The position of Apc11RING in the APC/C is more similar to Rbx1RING of activated cullin-Rbx1 structures.

a, Identification of Apc11 in apo APC/C. Left panel: EM density map for apo APC/C with the coordinates of Apc2CTD–Apc11 fitted (from APC/CCdh1.Emi1 structure). Right panel: EM density for APC/CApc11-ΔRING. The difference density corresponds to Apc11RING. EM density maps from ref. 4. b, Superimposed Apc2CTD onto Cul1CTD (PDB accession number 1LDK)61. c, Superimposed Apc2CTD onto Cul5CTD (PDB accession number 3DQV)27. In the inactive conformation of Cul1–Rbx1, Rbx1RING packs against WHB. In APC/CCdh1.Emi1 the location of Apc11RING remains in contact with Apc2CTD but has rotated 180° relative to inactive CRL structures, being similar to the swung out conformation of Rbx1RING of neddylated and activated Cul5–Rbx1 (ref. 27). d, e, The relative orientation of Apc2NTD and Apc2CTD is also dramatically different from Cul1 (ref. 61). This is due to a 70° rotation within cullin repeat 3 (between helices A–B and C–D–E), and a 20° rotation around the 4HB–cullin repeat 3 interface. Similar less pronounced structural variations are observed within the CRL family. d, Apc2–Apc11 (this study). e, Cul1–Rbx1 (PDB accession number 1LDK)61. f, The position of the Apc2CTD–Apc11 module differs slightly about the Apc2NTD–Apc2CTD interface between APC/CCdh1.Emi1 and APC/CCdh1.Hsl1.UbcH10–Ub.

Extended Data Figure 6 Three-dimensional classification of APC/CCdh1.Hsl1.UbcH10–Ub.

a, The three-dimensional classification (cycle 1) started with 477,850 motion-corrected particles, which were divided into ten classes. The resultant classes were grouped into five categories: (1) 58.8% in the active ternary state with coactivator and substrate (Hsl1); (2) 11.9% in the apo-inactive state; (3) 9.4% in a class with Cdh1 bound but with the catalytic module in the apo-inactive conformation (hybrid state); (4) 16.5% with weak Apc2 density; and (5) 3.4% were a poor reconstruction due to bad particles. Examination of the hybrid state (3) showed that density for Apc1WD40 was absent, explaining the lack of Cdh1-induced conformational change of the catalytic module. Particles in the ternary state (reconstructed to an overall resolution of 4.1 Å) were subjected to further three-dimensional classification (cycle 2). A major class (class 7, 26.4% of particles) showed improved density for UbcH10 (circled), and cycle 3 classification was performed on particles in this class. The major class of cycle 3 (class 5, 25.9% particles) showed further improved UbcH10 density. Further three-dimensional classification of this class did not improve the UbcH10 density. b, Particles in the best class (cycle 3, class 5, 19,939 particles) were refined in RELION and resulted in a map at 5.7 Å resolution (Extended Data Figure 2c). The UbcH10 density was improved by local alignment using a soft mask (indicated by circles) as described in Methods. c, Enlarged view of UbcH10 density. d, Enlarged view of the averaged APC/CCdh1.Hsl1.UbcH10–Ub reconstruction from cycle 1 of the three-dimensional classification (59% of particles). UbcH10 density is circled. e, Superimposition of classes 6, 7 and 8 of cycle 2 of the three-dimensional classification (from a), showing the structural variability of the three three-dimensional classes that indicate UbcH10 density. UbcH10 density is circled.

Extended Data Figure 7 Three-dimensional classification of APC/CCdh1.Hsl1.Apc11–UbcH10.

a, The three-dimensional classification started with 97,999 motion-corrected particles, which were divided into five classes. The resultant classes were grouped into three categories: (1) 80.6% in the active ternary state with coactivator and substrate (Hsl1); (2) 9.3% in the apo state; and (3) 10.1% in a hybrid state. Particles in the active ternary state (reconstructed to an overall resolution of 4.3 Å) were subjected to cycle 2 classification with ten classes. UbcH10 density in the resultant classes is indicated with circles. b, Classes with UbcH10 density in cycle 2 classification are superimposed, showing variability of UbcH10. c, Enlarged view of UbcH10 density. d, A negative-stain EM reconstruction of an APC/CCdh1.Hsl1 complex at 25 Å resolution with a 1,500-fold excess of UbcH10. The molecular surface is shown as a mesh representation and the coordinates of the APC/CCdh1.Hsl1.UbcH10–Ub were docked into the EM reconstruction. The UbcH10 coordinates fit new EM density proximal to Apc11.

Extended Data Table 1 EM data collection and processing statistics, and structure refinement statistics
Extended Data Table 2 Summary of model building of APC/C subunits
Extended Data Table 3 Table comparing APC/CCdh1.Hsl1 (ternary APC/C) and dissociation constants for UbcH10 (ref. 4) and UbcH10LR (this work).

Supplementary information

Video showing APC/CCdh1.Emi1 and APC/CCdh1.Hsl1.UbcH10-Ub reconstructions

Video showing overall architecture of the APC/C. Shown is a narrative of the structure starting with a view of the EM density map coloured-coded to represent the local resolution of the whole complex. The video shows the EM density map with the fitted atomic model represented as a cartoon and then atoms. The TPR lobe is shown coloured according to conservation (purple conserved) with TPR accessory subunits and the coactivator binding sites on Apc3, Apc8 and Apc1. Emi1 is shown interacting with Apc2-Apc11 and the structure of UbcH10 bound to Apc11 is shown with a substrate at the D-box site. (MOV 27050 kb)

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Chang, L., Zhang, Z., Yang, J. et al. Atomic structure of the APC/C and its mechanism of protein ubiquitination. Nature 522, 450–454 (2015). https://doi.org/10.1038/nature14471

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