The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of the G1 phase of the cell cycle. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C–substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-electron microscopy reconstruction of a human APC/C–coactivator–substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that the coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron-recognition module of coactivator and APC10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UBCH10–ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.
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We thank P. da Fonseca for her guidance preparing cryo electron microscopy grids and X. Bai and S. Scheres for help with the use of RELION and A. Plechanovova for advice in preparing stable UBCH10–Ub conjugates. We thank D. Morgan and W. Chao for their comments on the manuscript and D. Morgan for communicating data before publication. This work was funded by a Cancer Research UK grant to D.B.
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Extended data figures and tables
a, Coomassie blue stained SDS gel of the human APC/C–CDH1–HSL1 ternary complex. b, Silver stained SDS gel of apo APC/C and APC/CΔAPC15. c, Coomassie blue stained SDS gel of apo APC/C and APC/CΔRING. d, Cryo-electron microscopy micrograph of APC/C–CDH1–HSL1 ternary complex. e, Cryo-electron microscopy micrograph of apo APC/C (WT). f, Cryo-electron microscopy micrograph of APC/CΔRING. g, Cryo-electron microscopy micrograph of APC/CΔAPC15.
Extended Data Figure 2 Electron microscopy reconstructions of APC/C complexes described in this study.
a, Human APC/C–CDH1–HSL1 ternary complex. b, Apo APC/C. c, APC/CΔRING. d, APC/CΔAPC15. e, Fourier shell correlation (FSC) plots for four complexes. The resolution is based on the ‘Gold Standard’ criterion of an FSC at 0.143 (refs 54, 55). Also shown is the FSC plot between the atomic model and cryo electron microscopy map of APC/C–CDH1–HSL1.
a, Figure to show fitting of S. pombe APC6–APC12 (Cut9–Hcn26) crystal structure19 into the segmented APC6 electron microscopy density of APC/C–CDH1–HSL1. b, Superimposition of APC6–APC12 before (cyan) and after fitting using MDFF (pink). c, APC13 and APC16 interact with structurally equivalent sites (i) to (vii) on seven TPR subunits created by the TPR α-helices 8B, 9A and 9B. Shown are six sites (i) to (iii) (APC16) and (v) to (vii) (APC13). Roman numerals refer to interfaces labelled in Fig. 3e. d, Details of APC4WD40; e, APC1PC; f, APC1WD40. g, Electron microscopy density for CDH1; h, CDH1WD40 domain; i, APC10. j, APC10 interactions with APC1PC include the conserved 70s loop shown previously to mediate APC10 association with the APC/C32.
a, Selection of two dimensional class averages derived from negatively stained electron microscopy micrographs of APC4–mFab complexes. Two representative views of mFab are indicated by red arrows. b, Projections of the APC4 segmented density from the APC/C–CDH1–HSL1 cryo electron microscopy map match experimentally-derived two dimensional class averages of APC4.
a, The major classes were identified showing that the population consisted of 60% ternary complex and 40% apo complex (the CDH1 position is indicated with a red circle). b, Densities of APC2CTD–APC11 are visible only at lower contour level (outlined by a grey circle). Further three dimensional classification of the ternary complex into 10 classes revealed structural variability of the APC2CTD–APC11 module. In one class (Class 9), density for the entire APC2 is very weak, suggesting loss of this subunit in a small proportion (∼8%) of the total. c, Stereo-view of a superimposition of the 10 three dimensional classes. d, Although variable, APC2CTD–APC11 in the ternary complex shifts upward relative to the apo structure (yellow).
Extended Data Figure 6 APC/CCDH1 ubiquitination of HSL1 is reduced by APC10 and D box (P8 to P10) mutations.
Mutations of D box residues P8 to P10 to Ala (HSL1MutP8-10) and mutations of APC10 (S88A/N147A) (APC/CMutAPC10) at the D box-binding site reduce APC/C ubiquitination activity. Combining D box and APC10 mutations potentiated the reduction of APC/C ubiquitination activity. A contaminant band from CDH1 is indicated with an asterisk.
a, The 10 three dimensional classes of apo APC/C. b, Stereoview of a superimposition of the 10 three dimensional classes shows relatively little structural variability of the APC2CTD–APC11 module (circled).
Extended Data Figure 8 Comparison of APC/C–CDH1–HSL1 model with published APC/C electron microscopy reconstructions.
a, Human APC/C–CDH1–EMI1 (ref. 47). Density assigned to the inhibitory zinc-binding region and polybasic tail of EMI1 (EMI1ZT) is indicated. b, Human APC/CMCC (ref. 14). Density corresponding to the mitotic checkpoint complex (MCC) is indicated in a black border. This analysis shows that the activated conformation of APC/C observed in the APC/C–CDH1–HSL1 ternary complex is shared in the inhibited states of APC/C with EMI1 and the MCC (both with coactivator). The APC2CTD–APC11 module interacts with MCC and the inhibitory zinc-binding region (ZBR) and C-terminal polybasic tail of EMI1 (EMI1ZT).
a, SPR sensorgrams for ternary APC/C, apo APC/C and APC2CTD–APC11 binding to UBCH10, UBCH10–Ub, UBE2S and UBE2S-ΔC. b, Equilibrium fitting for ternary and apo APC/C association with UBCH10. The equilibrium dissociation constant (KeqD) is derived from this fit. c, Fit of the observed association rate constant (kobs) (kobs = kon[analyte] + koff) for ternary and apo APC/C association with UBCH10. d, Equilibrium fitting for ternary and apo APC/C association with UBCH10–Ub. e, Fit of the observed association rate constant (kobs) for ternary APC/C association with UBCH10–Ub. f, Equilibrium fitting for ternary and apo APC/C association with UBE2S. The reason for the lower maximum response unit (RU) at equilibrium for apo APC/C binding to UBE2S, relative to ternary APC/C, is unclear. g, Fit of the observed association rate constant (kobs) (for ternary and apo APC/C association with UBE2S. The rate for 865 nM apo APC/C was not fitted as this was close to the limit of the Biacore T200 response. h, Table summarizing APC/C–CDH1–HSL1 (ternary APC/C), apo APC/C and APC2CTD–APC11 dissociation constants for UBCH10, UBCH10–ubiquitin and UBE2S. KeqD, equilibrium dissociation constant; Kkind, kinetic dissociation constant = koff/kon. ND1, not determined because equilibrium binding was not achieved. ND2, not determined due to fast kon and koff. Standard errors of the fit are listed. ΔN-UBCH10 (deletion of residues 4–32 of UBCH10) and UBCH10 had similar relative affinities for ternary and apo APC/C (data not shown).
Video shows the structure of the human APC/CCdh1.Hsl1 ternary complex at 7.4 Å resolution. The molecular envelope colour-coded according to subunit assignments is shown. Second, the video shows the fitting of TPR subunit Apc6 and TPR accessory subunit Apc12 to EM density. The homo-dimers of Apc8, Apc6, Apc3 and Apc7 stack in parallel to form a left-handed supra-helix that maximises protein interfaces. Third, the video focuses on the platform subunits, Apc5, Apc15, Apc4, the Apc2-Apc11 catalytic module and Apc1. Fourth the degron recognition subunits Apc10 and Cdh1 together with EM density for the D box and KEN box are shown. (MP4 27490 kb)
Video morphs between apo APC/C and the APC/CCdh1.Hsl1 ternary complex, showing how the Cdh1 coactivator induces conformational changes of the platform subunits Apc2-Apc11, Apc4 and Apc5. The video shows morphing between the molecular envelopes followed by morphing between atomic models of the complex. Structures were first aligned using all subunits. Later, superimposing using the Apc1 PC domain as a reference indicates that the conformational change involves a rotation of the platform about an axis centred close to the Apc1PC-Apc8 interface. The insertion of Cdh1NTD at this interface disrupts Apc1PC-Apc8 interactions, shifting the platform subunits, displacing the Apc2CTD-Apc11 catalytic module. (MOV 7381 kb)
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Chang, L., Zhang, Z., Yang, J. et al. Molecular architecture and mechanism of the anaphase-promoting complex. Nature 513, 388–393 (2014). https://doi.org/10.1038/nature13543
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