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

Reviewer #1 (Public Review):

Summary:

This work focuses on the structure and regulation of the Anaphase-Promoting Complex/Cyclosome (APC/C), a large multi-subunit ubiquitin ligase that controls the onset of chromosome segregation in mitosis. Previous high-resolution structural studies have uncovered numerous structural features and regulatory mechanisms of the human APC/C, but it has remained unclear if these mechanisms are conserved in other model eukaryotes. To address this gap in our understanding, the authors employed cryo-electron microscopy to generate structural models of APC/C from the budding yeast S. cerevisiae, a key model organism in cell cycle analysis. In their comparison of the human and yeast complexes, the authors uncover many conserved structural features that are documented here in detail, revealing widespread similarities in the fundamental structural features of the enzyme. Interestingly, the authors also find evidence that two of the key mechanisms of human APC/C regulation are not conserved in the yeast enzyme. Specifically:

(1) The ubiquitin ligase activity of the APC/C depends on its association with a co-activator subunit such as CDH1 or CDC20, which serves both as a substrate-binding adaptor and as an activator of interactions with the E2 co-enzyme. Previous studies of the human APC/C revealed that co-activator binding induces a conformational change that enables E2 binding. In contrast, the current work shows that this E2-binding conformation already exists in the absence of a co-activator in the yeast enzyme, suggesting that the enhancement of E2 binding in yeast depends on other, as yet undiscovered, mechanisms.

(2) APC/C phosphorylation on multiple subunits is known to enhance APC/C activation by the CDC20 co-activator in mitosis. Previous studies showed that phosphorylation acts by promoting the displacement of an autoinhibitory loop that occupies part of the CDC20-binding site. In the yeast enzyme, however, there is no autoinhibitory loop in the CDC20-binding site, and there is no apparent effect of APC/C phosphorylation on co-activator binding sites. Thus, phosphorylation activates the yeast CDC20-APC/C by unknown mechanisms.

Strengths:

The strength of this paper is that it provides a comprehensive analysis of yeast APC/C structure and how it compares to previously determined human structures. The article systematically unwraps the key features of the structure in a subunit-by-subunit fashion, carefully revealing the key features that are the same or different in the two species. These descriptions are based on a thorough overview of past work in the field; indeed, this article serves as a concise review of the key features, conserved or otherwise, of APC/C structure and regulation.

Weaknesses:

No significant weaknesses were identified.

Reviewer #2 (Public Review):

Summary:

This paper from the Barford lab describes medium/high-resolution cryo-EM structures of three versions of the S. cerevisiae anaphase-promoting complex/cyclosome (APC/C):

(1) the recombinant apo complex purified from insect cells,

(2) the apo complex phosphorylated in vitro by cyclin-dependent kinase, and

(3) an active APC/C-Cdh1-substrate ternary complex.

The focus of the paper is on comparing similarities and differences between S. cerevisiae and human APC/C structures, mechanisms of activation by coactivator, and regulation by phosphorylation. The authors find that the overall structures of S. cerevisiae and human APC/C are remarkably similar, including the binding sites and orientation for the substrate-recruiting coactivator, Cdh1. In addition, the mechanism of Cdh1 inhibition by phosphorylation appears conserved across kingdoms. However, key differences were also observed that reveal divergence in APC/C mechanisms that are important for researchers in this field to know. Specifically, the mechanism of APC/C-Cdc20 activation by mitotic phosphorylation appears to be different, due to the absence of the key Apc1 autoinhibition loop in the S. cerevisiae complex. In addition, the conformational activation of human APC/C by coactivator binding was not observed in the S. cerevisiae complex, implying that stimulation of E2 binding must occur via a different mechanism in this species.

Strengths:

Consistent with the numerous prior cryo-EM structures of human APC/C from the Barford lab, the technical quality of the structure models is a major strength of this work. In addition, the detailed comparison of similarities and differences between the two species will be a very valuable resource for the scientific community. The manuscript is written very well and allows readers lacking expertise in cryo-EM to understand the important aspects of the conservation of APC/C structure and mechanism across kingdoms.

Weaknesses:

The lack of experimentation in this work to test some of the putative differences in APC/C mechanism (e.g. stimulation of E2 binding by coactivator and stimulation of activity by mitotic phosphorylation) could be considered a weakness. Nonetheless, the authors do a nice job explaining how the structure interpretations imply these differences likely exist, and this work sets the stage nicely for future studies to understand these differences at a mechanistic level. There is enough value in having the S. cerevisiae structure models and the comparison to the human structures, without any additional experimentation.

The validation of APC/C phosphorylation in the unphosphorylated and hyperphosphorylated states is not very robust. Given the lack of significant effects of phosphorylation on APC/C structure observed here (compared to the human complex), this becomes important. A list of phosphorylation sites identified by mass spec before and after in vitro phosphorylation is provided but lacks quantitative information. This list indicates that a significant number of phosphorylation sites are detected in the purified APC/C prior to reaction with purified kinases. Many more sites are detected after in vitro kinase reaction, but it isn't clear how extensively any of the sites are modified. There is reason for caution then, in accepting the conclusions that structures of unphosphorylated and hyperphosphorylated APC/C from S. cerevisiae are nearly identical.

Reviewer #3 (Public Review):

Vazquez-Fernandez et al. present a comprehensive and detailed analysis of the S. cerevisiae APC/C complex, providing new insights into its structure and function. The authors determined the medium-resolution structures of three recombinant S. cerevisiae APC/C complexes, including unphosphorylated apo-APC/C (4.9 Å), the ternary APC/CCDH1-substrate complex (APC/CCDH1:Hsl1 , 4.0 Å), and phosphorylated apo-APC/C (4.4 Å). Prior structures of human, E. cuniculi, S. cerevisiae, and S. pombe APC/C subunits, as well as AlphaFold2 predictions were used to guide model building. Although the determined structures are not sufficient to fully explain the molecular mechanism of APC/C activation and regulation in S. cerevisiae, they provide valuable insights into the similarities and differences with the human complex, shedding light on the conserved and divergent features of APC/C function.

The manuscript synthesizes the structural analysis of the APC/C complex in S. cerevisiae, with literature into a cohesive and clear picture of the complex's structure and function. It is well-written and clear, making the complex biology of the APC/C complex accessible to a wide range of readers. The complex forms a triangular shape, with a central cavity surrounded by two modules: the TPR lobe and the platform module. The TPR lobe consists of three TPR proteins (APC3, APC6, and APC8), which stack on top of each other to form a quasi-symmetric structure. The platform module is composed of the large APC1 subunit, together with APC4 and APC5. The authors also analyzed the structure of several smaller subunits that are involved in regulating the activity of the APC/C complex and showed their structural similarities to and discrepancies from their human counterparts. These subunits, including CDC26/APC12, SWM1/APC13, APC9, and MND2/APC15, form extended, irregular structures that simultaneously contact multiple large globular APC/C subunits.

While the authors report the similarity between the overall structure of S. cerevisiae and human APC/C complexes, they also found two unexpected differences. First, in the S. cerevisiae apo-complex, the E2 binding site on APC11RING is accessible, whereas, in humans, it requires CDH1 binding. Second, a structural element similar to the human APC1 auto-inhibitory segment is missing in S. cerevisiae. In humans, the phosphorylation-dependent displacement of this segment allows CDC20 binding to APC/C. In S. cerevisiae, the binding requires phosphorylation however the structures reported here are suggestive that this could involve a different (presently unknown) mechanism. These structural insights highlight the importance of understanding the species-specific features of APC/C function.

Strengths:

The manuscript does a great job of revealing new structures.

Opportunity for increasing impact: It would have been nice if some functional differences were demonstrated, for example regarding the mechanism of CDC20 binding, and the comparison between apo-APC/C and ternary APC/CCDH1:Hsl1 does not explain the molecular activation mechanism of S. cerevisiae APC/C. Nonetheless, the authors nicely integrate their data with well-established literature on the similarities and differences between yeast and human systems.

Weaknesses:

The authors should cite and discuss Cole Ferguson et al., Mol Cell 2022. This study describes the loss of APC7 in human disease and provides a detailed structural and biochemical examination of the effects of APC7 loss on human APC/C. Given that much of our understanding of APC7 comes from this work, it should be highlighted in the introduction and discussed in depth in light of the new work on S. cerevisiae APC/C.