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  • While SCF E ligase activity was reconstituted with recombina


    While SCF E3 ligase activity was reconstituted with recombinant proteins two decades ago, the ability to probe APC/C was limited until recently because of its behemoth size. Human APC/C is a 1.2-MDa assembly comprised of 19 core subunits (one each of nine different APC subunits, and two each of five), which catalyzes ubiquitylation in collaboration with an additional coactivator protein and a Ub-linked E2 conjugating enzyme (Figure 1A and Box 1) (reviewed in 13, 14, 15). The variable substrate receptors are CDC20 and CDH1, which are termed coactivators due to their successively activating APC/C during mitosis by both recruiting substrates 16, 17, 18 and conformationally activating the catalytic core 19, 20, 21 (Figure 1). The catalytic core consists of the cullin and RING subunits APC2 and APC11 22, 23, 24. The APC2–APC11 cullin–RING assembly directs Ub transfer from an amitriptyline hydrochloride of E2 enzymes with different specificities 25, 26, 27. Repeated cycles of Ub transfer lead to polyubiquitylation, wherein multiple individual Ubs become linked to the substrate and to each other to form Ub chains. There is enormous diversity in the architecture of potential Ub chains produced by APC/C, with the number of Ubs, and the sites of their chain linkages, thought to influence the rates of substrate degradation by the proteasome. The E2 enzyme UBE2C/UBCH10 (or in some circumstances the E2 UBE2D/UBCH5 [28]) directly modifies substrates with one or more Ubs or short Ub chains (reviewed in 13, 14, 15), which are sufficient to target some human APC/C substrates for degradation [29]. However, many substrates are degraded after a different E2 enzyme, UBE2S in humans 30, 31, 32, extends a poly-Ub chain. Ub is transferred from the catalytic cysteine UBE2S to Lys11 on a Ub that is already attached to a substrate. Often, branched chains are formed when UBE2C first modifies a substrate with Lys48-linked Ub chains, and then UBE2S further extends these chains with additional Ubs connected via Lys11. These Lys48/Lys11 branched chains are particularly potent at directing substrates for proteasomal degradation [33]. How does APC/C recognize its substrates and catalyze their ubiquitylation? How are the outcomes and timing of these activities regulated? These questions have driven a decade of structural studies that begin to explain how APC/C interacts with coactivators, substrates, and E2s, and how these interactions are tightly controlled by phosphorylation and inhibitory proteins to prevent premature cell division and collateral catastrophic consequences like aneuploidy (reviewed in 13, 14). Detailed insights into the stepwise regulation of APC/C throughout the cell cycle have come in the last few years from advances in generating recombinant multiprotein complexes and cryo-electron microscopy (cryo-EM). Structural details provided by cryo-EM reconstructions of APC/C complexes, and X-ray crystallography and NMR data on subcomplexes, as well as enzymology of ubiquitylation by wild-type and mutant versions of recombinant APC/C, have been described in recent excellent reviews 15, 34. However, we now understand that APC/C undergoes striking conformational changes accompanying its assembly into distinct complexes, as many recent structural studies have also shown that the ability of APC/C to perform different activities depends on rotation of coactivators, and on cullin and RING subunits transitioning from an intertwined inactive unit into conformationally mobile appendages 19, 20, 21, 24 that bind and are harnessed by different substrates, E2s, and inhibitors into distinct conformations specifying particular functions. Here, we summarize overall structural features of human APC/C, focusing on the emerging understanding of how its different conformations are achieved to establish regulation.
    Overall APC/C Organization Early EM and other studies of APC/C from several organisms revealed that APC/C is formed from modules that together adopt an overall curved structure around a central cavity displaying flexibly tethered protein binding domains 24, 35, 36, 37, 38, 39, 40, 41, 42, 43. These flexible domains recruit, and in turn are positioned by, APC/C’s many binding partners including substrates and the E2 enzymes that modify them (Figure 1 and Box 1). This architecture both juxtaposes the binding partners of APC/C, and enables a remarkable spectrum of conformations establishing the functions of this E3 ligase.