• 2018-07
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  • 2020-01
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  • 2020-10
  • The structure of the HOIP


    The structure of the HOIP RBR–LDD module bound to UbcH5~Ub reveals insights into an activated HOIP component [47]. Contacts between the E2~Ub and the non-cognate RBR module in the crystal suggest how a structure in which the active sites of the E2 and the E3 are in close proximity might look. In this complex, the E2 binds to RING1 and the Talabostat mesylate sale surface of ~Ub exposed in the open E2~Ub contacts the IBR–RING2 linker, bringing RING2 close to the E2 (Fig. 4B). For these contacts to be made by the cognate HOIP chain, other contacts involving the IBR will have to be disrupted. Finally, comparison of the HOIP RBR–LDD/E2~Ub and the RING2–LDD~Ub/Ubacceptor complex structures reveals that prior to the formation of the RING2~Ub species, the Ub moiety of E2~Ub bound to RING1 resides at the same RING2 site as the acceptor Ub that is required for Ub chain formation [40], [47]. This observation seems to imply that Ub transfer from the E2~Ub onto the E3 active site must occur before the substrate (acceptor Ub) can bind in a manner that poises it for the ultimate reaction. There is currently no evidence whether a similar order of events is shared by other RBR E3s.
    Concluding remarks While the last to be identified as a mechanistic class of E3 ligases, research on RBR E3s has provided important insights in a relatively short time frame. The main conceptual framework is in place: Important and fundamental questions remain:
    Discussion We conclude that the binding of activator to the APC/C enhances E2 efficiency by multiple mechanisms. These mechanisms are reminiscent of the effects of neddylation on SCF activity [18, 19] and might therefore reveal conserved general mechanisms in ubiquitin ligase regulation. The dramatic decrease in the Km for E2 upon activator binding suggests that the major effect of activator is to increase the affinity of the interaction between APC/C and E2-Ub. The relatively small increase in APC/C turnover rate at saturating E2 concentrations is less easily explained, but it could result from changes in E2-Ub orientation or positioning near the substrate [3, 18, 19] or from some change in the ability of the RING domain to stimulate E2 activity [24, 25, 26]. How might the activator elicit these effects? Previous studies in Xenopus extracts suggest that the N-terminal region of the activator is responsible for promoting ubiquitination of a substrate, Nek2A, that associates with the APC/C in the absence of activator [6]. Consistent with these studies, we found that the C box of the N-terminal region is required for the stimulatory effect of activator. Interestingly, high-resolution electron microscopy studies of human APC/C structures have recently revealed that the activator N-terminal region promotes a major shift in the positioning of the E2-binding site formed from the cullin and RING subunits (D. Barford, personal communication). These conformational changes are consistent with the shifts in E2 biochemical behavior that we observe in the presence of activator. We observed that mutation of substrate degrons greatly reduces the impact of activator on the Km for E2, suggesting that degron binding promotes an increase in E2 affinity. How might this occur? Perhaps substrate binding to the activator C-terminal WD40 domain influences the ability of the activator N-terminal region to exert its effects on the E2-binding site. Given that substrate enhances activator binding to the APC/C [27], one possibility is that the fusion substrate locks down the WD40 domain on the APC/C, leading to higher-affinity binding by the N-terminal region. It seems likely that substrate degron binding also influences activity through E2-independent mechanisms. Interaction of the fusion substrate with activator should restrain the mobility of the large substrate N-terminal region, promoting more productive substrate positioning and thereby enhancing catalytic rate. In addition, substrate-activator interactions might affect the accessibility of certain lysines, as suggested by the changes in patterns of lysine modification in reactions with some degron mutants (Figures 1E and 1F). Interestingly, in the case of securin at least, the D box stimulated fusion substrate ubiquitination even in the absence of activator (Figure 1E; Table S1), perhaps as a result of the low-affinity interaction between the D box and Apc10 alone [12].