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  • One architecture termed closed has been visualized at near

    2020-11-20

    One architecture, termed closed, has been visualized at near-atomic resolution in cryo-EM maps of recombinant complexes that superimpose with earlier lower-resolution EM data obtained for APC/CCDC20–MCC purified from HeLa Sunitinib Malate arrested during the mitotic checkpoint (Figure 4E) 38, 48, 51. Here, MCC essentially fills the entire central cavity, and blocks substrate-binding sites by enwrapping CDC20A through multiple elements, including a D box, an ABBA motif, and a KEN box 48, 51, 82, 83. In addition, residues proximal to the CDC20M propeller interact with CDC20A adjacent to its D-box binding site. The closed configuration is secured by interactions involving multiple distal MCC elements 48, 51. First, the IR tail from the CDC20M of MCC docks in the vacant TPR groove from the second APC8 molecule in APC/C. Second, the BUBR1 subunit of MCC hijacks the UBE2C-binding surface on the WHB subdomain of APC2. As with the APC/C conformations that mediate ubiquitylation, this inhibited architecture depends on mobility of the WHB domain, which underlies its capture in different orientations by MCC and UBE2C. Because there is no sequence or structural homology between BUBR1 and UBE2C, this interaction was unexpected and demonstrated multifunctionality of a WHB domain of cullin. Unlike EMI1, MCC does not hijack any known elements for UBE2S-dependent Ub chain elongation, explaining how APC/CCDC20-MCC complexes remain competent for UBE2S-dependent free Ub chain elongation 48, 51, 75. Nonetheless, it remains unknown whether this UBE2S activity is irrelevant due to the absence of UBE2C-dependent substrate ubiquitylation, or whether there is functional importance for MCC-bound APC/CCDC20 in extending poly-Ub chains.
    Freeing APC/C from Inhibitors for Ub-Dependent Cell Division In order for cell division to proceed, APC/C must be liberated from inhibitors to catalyze UB-dependent turnover of its substrates. EMI1 is removed and degraded by several phosphorylation and ubiquitylation-dependent mechanisms 84, 85 that may vary in detail among organisms [86] and that remain mechanistically perplexing [87] in part due to lack of structural details. However, structural mechanisms contributing to liberation of APC/CCDC20 from MCC have recently been elucidated. When the spindle assembly checkpoint is satisfied, chromosomes are correctly aligned on the mitotic spindle, cells are prepared for anaphase, and MCC dissociates from APC/CCDC20 in a manner that involves UBE2C-dependent ubiquitylation of the CDC20M subunit within MCC 56, 57. Structural reconstitution of this reaction reveals massive conformational changes 48, 51. In addition to the closed conformation of APC/CCDC20–MCC described above that excludes UBE2C, cryo-EM structures also reveal more open configurations (Figure 4F) 48, 51. Here, MCC still blocks canonical substrate-binding sites on CDC20A, but the CDC20A–MCC portion of the complex is rotated out of the APC/C central cavity. Concomitantly, the APC2–APC11 cullin–RING catalytic core is free of MCC, conformationally activated, and available (Figures 2 B and 4 F). Additional low-resolution EM structures show that within an open configuration of APC/CCDC20–MCC, UBE2C is placed by both the APC2 WHB domain and APC11 RING domain adjacent to the MCC target lysines (Figures 2 B and 4 G) 48, 51, 88, which would drive Ub-dependent regulation of APC/CCDC20–MCC dissociation. The question arises as to how the massive conformational changes determining whether APC/CCDC20–MCC is inhibited or able to catalyze MCC ubiquitylation are naturally controlled? This transition likely involves APC/C regions near the subunit APC15, because reducing cellular APC15 levels stabilizes MCC on APC/C 88, 89, 90. Indeed, recombinant APC/CCDC20–MCC complexes lacking APC15 preferentially adopt the closed conformation that inhibits UBE2C-dependent ubiquitylation 48, 51, although APC15 is not required for APC/CCDC20–MCC to swing away from the APC2–APC11 catalytic core 48, 51, nor is there evidence that APC15 ever cycles on and off APC/C. It is possible that all APC/CCDC20–MCC complexes continually cycle between the closed and open states. However, differences between the structural studies indicate a role for phosphoregulation: different ratios of open versus closed configurations of recombinant APC/CCDC20–MCC are observed 48, 51, depending on whether the APC/CCDC20 accumulates phosphorylation during expression in insect cells [53], or whether potential phosphorylation is mimicked by glutamate replacements for 100 possible mitotic phosphorylation sites [54]. The two preparations likely differ in terms of where negative charges are placed, raising the possibility that certain negatively charged phosphorylation sites may modulate the conformation of APC/CCDC20–MCC in vivo to regulate termination of the spindle assembly checkpoint.