br Coactivator Binding Sets APC C Catalytic Core in Motion
Coactivator Binding Sets APC/C Catalytic Core in Motion A coactivator not only recruits substrates to APC/C  (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  (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 . 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 EVP4593 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 . Positioning substrate lysines proximal to an activated E2∼Ub intermediate is therefore crucial for ubiquitylation. The best-characterized APC/C substrates are recruited via various linear motifs (e.g., D boxes, KEN boxes, and ABBA motifs) that serve as degrons, binding to distinct regions of the β-propeller domain of a coactivator (Figure 1, Figure 3B) (reviewed in ). In addition, D boxes also simultaneously bind the APC/C core subunit APC10, thereby fastening the coactivator propeller adjacent to APC10 (Figures 2 A and 3 B) 39, 41, 45, 47. The positions of potential target lysines within substrates are thus determined by their locations relative to degrons bound to an APC/C–coactivator complex, and the location of the coactivator propeller determined by the presence or absence of a D box. The E2 UBE2C (also known as UBCH10) is recruited to APC/C by a specialized mechanism that places the active site proximal to substrates (Figure 4B). One side of the UBE2C∼Ub intermediate engages the RING domain of APC11 through canonical RING-E2∼Ub interactions 47, 49. However, as in other cullin–RING ligases , the RING domain of APC11 is loosely tethered to the cullin-binding region by a flexible linker that rotates (Figure 4). How then, does the RING-bound UBE2C∼Ub intermediate achieve a position with the active site facing substrates? Unexpectedly, the flexibly tethered cullin element at the C terminus of APC2 – the WHB subdomain – binds the so-called E2 backside of UBE2C distal from the active site (Figure 2B) . Thus, the two flexibly tethered domains of APC11 and APC2 together grasp opposite sides of UBE2C, acting like a clamp to direct the catalytic center toward substrates . This structural arrangement provides a potential rationale for why many APC/C substrates are ubiquitylated in intrinsically disordered regions: flexible polypeptides bound to a coactivator can access the relatively proximal but immobilized UBE2C active site 47, 49, 50, 69. Indeed, the disordered N-terminal domain of cyclin B can receive enough individual Ubs from UBE2C for proteasomal targeting even without generation of poly-Ub chains . The confined space between the active site of UBE2C and a coactivator-bound degron may also explain why UBE2C preferentially modifies substrates with individual Ubs and short chains rather than long poly-Ub chains .