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  • CAL-101 what Across the scaffold the platform anchors


    Across the scaffold, the platform anchors the N terminus of the elongated cullin structure of APC2 [21]. This connects to the flexible cullin–RING catalytic core consisting of the C-terminal region of APC2 and the associated APC11 11, 12, 21. The flexibility and positions of the catalytic core are controlled by the orientation of the platform, and by proteins interacting directly with APC2–APC11 to regulate ubiquitylation 21, 24, 47, 48, 49, 50, 51, 52.
    Visualizing APC/C Interactions and Functions around the Cell Cycle Throughout the cell cycle, APC/C undergoes a series of transformations between assemblies for which cryo-EM data revealed that functions are dictated in part by conformations of the coactivator and the cullin–RING catalytic core. By the end of interphase, APC/C is hypophosphorylated, coactivator free, and inactive, in part due to autoinhibition, whereby intramolecular interactions block access of CDC20 and restrain the cullin–RING catalytic core 21, 24, 53, 54, 55 (Figure 2A, Conformation I). In prophase, phosphorylation conformationally activates APC/C binding to the coactivator CDC20, which in turn conformationally activates the catalytic core (see Video S1 in the Supplemental information online) 21, 24, 53, 54, 55. APC/CCDC20 is, in principle, competent to recruit substrates for ubiquitylation 21, 24 (Figure 2A, Conformation IIA). However, the timing of substrate binding is regulated by the mitotic checkpoint complex (MCC), which serves as a brake during the spindle assembly checkpoint when APC/C has been activated by CDC20 but cells are not yet prepared for division 48, 51 (Figure 2B, Conformation III). When all chromosomes are properly bioriented on the mitotic spindle, both MCC and the catalytic core are reoriented (Figure 2B, Conformation IV) so MCC can itself be ubiquitylated as a prelude to liberating APC/C for substrate ubiquitylation that triggers CAL-101 what 48, 51, 56, 57 (Figure 2B, Conformation V). In anaphase, APC/CCDC20 recruits substrates, while the APC2–APC11 cullin–RING catalytic core engages, positions, and activates transient E2∼Ub intermediates (e.g., UBE2C∼Ub, where ‘∼’ refers to thioester bond) from which Ub is transferred to a coactivator-bound substrate (Figure 2B, Conformation VI) or to a substrate-linked Ub molecule during polyubiquitylation (Figure 2B, conformation VII) 47, 49, 50, 53. Following anaphase, CDC20 itself undergoes APC/C-mediated ubiquitylation and subsequent degradation, but is replaced by the homologous but distinct coactivator CDH1 that recruits other substrates for ubiquitylation (Figure 2A, Conformation IIB) 47, 49, 51. After substrates are degraded, then EMI1 inhibits APC/CCDH147, 58, 59, 60 (Figure 2B, Conformation VIII) to enable accumulation of G1 cyclins and CDK-dependent inactivation of CDH1.
    Conformational Activation of APC/C Enables Binding to the Coactivator CDC20 APC/C comes to life by binding a coactivator. This is controlled in part by phosphorylation and APC/C conformational changes that expose the binding sites for the C box and IR tail of the coactivator. In interphase when mitotic kinase activity is low, CDC20 binding is blocked 61, 62, 63. A recent cryo-EM structure indicates that in the absence of phosphorylation, an APC1 loop occupies the C-box-binding site of APC8 [53] (Figure 3A). Contemporaneous biochemical studies using recombinant APC/C have shown the mechanism by which mitotic phosphorylation relieves this inhibition to permit CDC20 binding: whereas unphosphorylated serines in the APC1 loop engage the CDC20-binding site by binding proximal to acidic surfaces, their phosphorylation during the cell cycle – or substitution with phosphomimicking glutamate mutations or deletion in recombinant APC/C – prevents the autoinhibitory interaction and frees the APC8 groove to bind the C box of CDC20 53, 54, 55. Structural studies of the other coactivator-binding site – the APC3 groove recruiting the IR tail of CDC20 or CDH1 – also raise the possibility of conformational control (Figure 3A). Two conformations have been characterized, an open form with the C-terminal TPR superhelical groove exposed to engage an IR tail (Figure 3A), and a closed form in which the C-terminal helices of APC3 are dramatically rearranged to pack in the groove (Figure 3B) 21, 47, 64. Both conformations were observed in apo-APC/C [53]. Although it remains unknown if the APC3 conformations are simply in equilibrium or if APC3 binding to the IR-tail of a coactivator is regulated, it is conceivable that docking of the C box of a coactivator in the APC8 groove could potentially trigger conformational changes throughout the TPR lobe that influence opening of the APC3 groove.