Chromosomes and Gene Expression

A comparative study of the cryo-EM structures of S. cerevisiae and human anaphase-promoting complex/cyclosome (APC/C)

Ester Vazquez-Fernandez
Jing Yang
Ziguo Zhang
Antonina E Andreeva
Paul Emsley
David Barford author has email address

MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK

https://doi.org/10.7554/eLife.100821.1

Open access
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Figures and data

Overall structure of the APC/C^CDH1:Hsl1 complex. a, Two views of the APC/C^CDH1:Hsl1 ternary complex fitted into the 4.0 Å cryo-EM map. b, Two views of the APC/C^CDH1:Hsl1 ternary complex shown as ribbon representations.

Overall structure of unphosphorylated apo-APC/C. a, Two views of apo-APC/C fitted into the 4.9 Å cryo-EM map. b, Two views of apo-APC/C shown as ribbon representations.

Overall structure of phosphorylated apo-APC/C. a, Two views of phosphorylated apo- APC/C fitted into the 4.5 Å cryo-EM map. b, Two views of apo-APC/C shown as ribbon representations.

Comparison of S. cerevisiae APC/C^CDH1:Hsl1 with human APC/C^CDH1:Emi1. a, S. cerevisiae and b, human complexes subunits colour-coded. c, S. cerevisiae and d, human complexes with large subunits coloured in grey and small IDP subunits (APC9, APC12, APC13, APC15, APC16) colour-coded. The structure of human APC/C^CDH1:Emi1 from (Höfler, Yu et al. 2024) (PDB 7QE7). e, and f, Four small IDP-subunits (APC9, APC13, APC15, APC16) contribute to interacting with equivalent, quasi-symmetrical sites on the outer surfaces of the TPR subunits of the TPR lobe. TPR lobes of S. cerevisiae (e) and human APC/C (f) are depicted as a surface representations. The contact sites with the three small subunits that contact the S. cerevisiae TPR lobe are numbered 3 to 8 after human APC/C (Chang, Zhang et al. 2015) that has sites 8 sites sue to APC7.

Comparison of S. cerevisiae and human APC2 and APC4 subunits. a,
Human APC2 with APC2^ZBM of CRL2 coloured red. b, S. cerevisiae APC2. The position of human APC2^ZBM is indicated. c, Superimposition S. cerevisiae APC2 (purple) with human APC2 (grey) on CRL2. Human APC2 from (Höfler, Yu et al. 2024) (PDB 7QE7). d, S. cerevisiae APC4, colour-coded from N- to C- termini with a blue to red ramp. e, Human APC4, colour-coded from N- to C-termini with a blue to red ramp. S. cerevisiae APC4^WD40 is a six-bladed β-propeller in contrast to the seven bladed β-propeller of human APC4^WD40. f, AlphaFold2 prediction of S. cerevisiae APC4 colour-coded according to pLDDT score. No seventh bade is predicted. The α-helix in place of the seventh blade is predicted with low confidence. g, Fit of S. cerevisiae APC4^WD40 into the APC/C^CDH1:Hsl1 cryo-EM map showing lack of density for a seventh blade. Human APC4 from (Höfler, Yu et al. 2024) (PDB 7QE7).

CDH1^NTD contacts to APC/C and control by phosphorylation. a, In S. cerevisiae CDH1, α2, α3 and α6 contact the PC domain of APC1 (APC1^PC) (conserved with human CDH1). α2 also contacts APC8B. α5, unique to S. cerevisiae CDH1, forms extensive contacts to APC6B. Human CDC20^NTD (light green) shares α2 and α3 with CDH1^NTD. b, MSA of S. cerevisiae and human CDH1^NTD and CDC20^NTD showing α-helices α2 to α6 of S. cerevisiae CDH1^NTD and the conserved sites of CDK phosphorylation on CDH1 (red arrows). c, S. cerevisiae CDH1^NTD colour-coded from N- to C-termini with a blue to red ramp interacting with APC1^PC, APC6B and APC8B. d, Same view as (c) without APC1^PC, APC6B and APC8B. e, Superimposition of S. cerevisiae CDH1^NTD coloured blue-to- red, human CDH1^NTD (grey) (from (Höfler, Yu et al. 2024), PDB 7QE7) and human CDC20^NTD (light green) (from (Zhang, Tischer et al. 2019) PDB 6Q6G). α-helices α2, α3 and α6 are conserved in all three structures.

Comparison of CDH1^IR binding to S. cerevisiae and human APC/C^CDH1 complexes. a,
Overall view of the CDH1^IR-binding site on S. cerevisiae APC/C^CDH1:Hsl1. b, Zoomed view of this complex. c, Overall view of the CDH1^IR-binding site on human APC/C^CDH1:Emi1. d, Zoomed view of this complex. The structure of human APC/C^CDH1:Emi1 from (Höfler, Yu et al. 2024) (PDB 7QE7). e, f, APC9 of S. cerevisiae APC/C partially mimics APC7 and APC16 of human APC/C. (e) In S. cerevisiae APC/C, the interaction of APC9 with APC3A (segment 2 of APC9) and APC3B (segment 3 of APC9) partially mimics the respective APC7 and APC16 interactions with human APC3. APC9 segment 2 interactions with the C-terminal TPR helix of APC3A may stabilise the CDH1^IR-binding site of APC3A. f, Similarly, in human APC/C, the interface of APC7B with APC3B might function to stabilise the APC10^IR-binding site on APC3B. Human APC/C^CDH1:Emi1 from (Höfler, Yu et al. 2024) (PDB 7QE7).

Comparing apo-APC/C and the APC/C^CDH1:Hsl1 complex shows both apo-APC/C and the APC/C^CDH1:Hsl1 complexes are competent to bind the Ubc4. a, Two views of apo-APC/C. b, Two views of APC/C^CDH1:Hsl1. The coordinates were superimposed on APC1^PC. In the apo-APC/C complex, there are no contacts between APC1^PC of the platform module and APC6B and APC8B of the TPR lobe. In the APC/C^CDH1:Hsl1, CDH1^NTD bridges APC1^PC with APC6B and APC8B. The overall conformations are similar, specifically the APC2:APC11 catalytic module adopts a raised conformation in both states. c, Model of APC/C^CDH1:Hsl1 in complex with Ubc4. d, Model of apo-APC/C in complex with Ubc4. The position of the APC2:APC11 catalytic module in apo-APC/C does not exclude Ubc4 binding (d). Model of APC11:E2 based on the S. cerevisiae Not4 RING:Ubc4 complex (PDB 5AIE) (Bhaskar, Basquin et al. 2015).

Regulation of APC/C by phosphorylation. The C-box binding site is un-obstructed in unphosphorylated apo-APC/C. a, Cryo-EM density from the unphosphorylated apo-APC/C map corresponding to the C-box binding site of APC8B with the fitted CDH1^C-box modelled from APC/C^CDH1:Hsl1. We observe no density at the APC8B C-box binding site. b, Cryo-EM map density for the CDH1^C-box from the APC/C^CDH1:Hsl1 reconstruction with fitted coordinates. c-e, Comparison of unphosphorylated apo-APC/C and phosphorylated apo-APC/C: (c) Unphosphorylated apo-APC/C. (d) Phosphorylated apo-APC/C. (e) Superimposition of unphosphorylated apo-APC/C (coloured by subunit assignment) and phosphorylated apo-APC/C (grey). There is a small relative shift of the TPR lobe. The coordinates were superimposed on APC1^PC.

SDS PAGE gels of purified APC/C complexes and ubiquitylation assays. a, APC/C^CDH1:Hsl1 complex, unphosphorylated apo-APC, phosphorylated apo-APC (lanes 1- 3). b, Ubiquitylation assay showing activation by CDH1. Phosphorylation (P: phosphorylated APC/C) did not significantly affect activity. c, Time course of ubiquitylation reaction.

EM images and 2D class averages of APC/C^CDH1:Hsl1 complex. a, Representative negative stain image of the APC/C^CDH1:Hsl1 complex. b, Representative cryo-EM image of the APC/C^CDH1:Hsl1 complex. c, Gallery of 2D-class averages of APC/C^CDH1:Hsl1. d, Fourier Shell Correlation (FSC) plots of the main maps used to generate molecular models APC/C^CDH1:Hsl1, unphosphorylated apo-APC/C and phosphorylated apo-APC/C. e, Local resolution map of APC/C^CDH1:Hsl1. f, Fourier Shell Correlation (FSC) plots of the main maps used to generate molecular models.

Cryo-EM images and 2D class averages of apo-APC/C complexes. a,
Representative cryo-EM image of unphosphorylated apo-APC/C. b, Gallery of 2D-class averages of unphosphorylated apo-APC/C. c, Representative cryo-EM image of phosphorylated apo-APC/C. d, Gallery of 2D-class averages of phosphorylated apo-APC/C.

Data processing pipeline for APC/C^CDH1:Hsl1 complex cryo-EM reconstructions. a,
Cryo-EM data processing workflow summary for the APC/C^CDH1:Hsl1 complex as described in the Methods section. b, Example of cryo-EM map with fitted coordinates.

Data processing pipeline for unphosphorylated apo-APC/C cryo-EM reconstructions.
Cryo-EM data processing workflow summary for unphosphorylated apo-APC/C as described in the Methods section.

Data processing pipeline for phosphorylated apo-APC/C cryo-EM reconstructions.
Cryo-EM data processing workflow summary for phosphorylated apo-APC/C as described in the Methods section.

Conformational change of APC1 on conversion from apo-APC/C to APC/C^CDH1:Hsl1. a, Superimposed on APC1^PC. b, Superimposed on the combined APC1^Mid-APC1^WD40 domains. On conversion, there is a small tilt of APC1^PC relative to the combined APC1^Mid-APC1^WD40 domains that form a rigid unit.

Summary of CDK phosphorylation sites of in vitro phosphorylated apo-APC/C identified by mass spectrometry

Summary of CDK phosphorylation sites of in vitro phosphorylated apo-APC/C identified by mass spectrometry

Cryo-EM data collection, refinement and validation statistics.

Cryo-EM data collection, refinement and validation statistics.

Ordered and disordered regions of APC/C^CDH1:Hsl1 subunits

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