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  • br Conformational Activation of APC C Enables Binding to

    2019-07-02


    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 IOX2 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 IOX2 changes throughout the TPR lobe that influence opening of the APC3 groove.
    Coactivator Binding Sets APC/C Catalytic Core in Motion A coactivator not only recruits substrates to APC/C [18] (Figure 3B), but also stimulates repositioning of the catalytic core 19, 24. High-resolution cryo-EM maps of apo forms of APC/C without a coactivator show the catalytic core and platform rigidified from the APC2–APC11 α/β domain and RING domain straddling the APC4 helical domain [53] (Figures 1C, 2A, and 4A,Box 1, and Video S1 in Supplemental information online). This down conformation blocks the canonical E2-binding site on the RING domain of APC11, and renders the central cavity wide open, presumably for access to activating kinases and coactivators. Coactivator binding induces substantial APC/C conformational changes that globally shift the platform and catalytic core into proximity of the substrate-binding module and increase the mobility of domains contributing to catalysis 21, 24 (Figures 2 A and 4 B). Most strikingly, repositioning of APC4 eliminates contacts with the catalytic core observed in apo-APC/C [47]. The liberated C-terminal domain of APC2 and the associated APC11 RING domain become mobile in an activated up location as indicated by their low resolution in EM maps of APC/C–coactivator–substrate complexes (Figure 2A) 21, 24, 39, 41, 53. Indeed, much regulation of APC/CCDC20 and APC/CCDH1 depends on various partner proteins harnessing different binding sites on the flexibly tethered coactivator and cullin and RING portions of the catalytic core.
    Harnessing the Mobile APC/C for Initial Ub Transfer Directly onto Substrates For ubiquitiylation to occur, substrates and Ub carrying enzymes must be juxtaposed. Furthermore, RING E3s typically activate the ligation reaction, that is, Ub transfer, by the RING domain binding both the E2 catalytic domain and its thioester-bonded Ub. This stabilizes weak interactions between the E2 and Ub in a closed conformation 65, 66, 67 that strains and stimulates reactivity of the thioester bond between them [68]. Positioning substrate lysines proximal to an activated E2∼Ub intermediate is therefore crucial for ubiquitylation.