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  • br Ubiquitin Capture by the Mobile APC RING Domain

    2019-08-29


    Ubiquitin Capture by the Mobile APC11 RING Domain Contributes to Processive Substrate Ubiquitylation Although the rules of Ub-mediated proteolysis are only beginning to emerge, the rate and order in which different APC/C substrates are degraded during the Cy7.5 NHS ester (non-sulfonated) correlates with processivity of their ubiquitylation. Highly processive substrates receive enough Ubs in a single binding event to enable proteasome binding, and are apparently degraded earlier during the cell cycle than nonprocessive substrates that must cycle on and off APC/C numerous times to receive enough Ubs for proteasomal targeting [71]. Degradation of less-processive substrates can be further slowed by competition with other substrates preventing their rebinding to APC/C, and deubiquitylation removing the few initially linked Ubs [72]. Processivity is determined in part by the rate of dissociation of a substrate from APC/C, which depends on the affinity and arrangement of degron sequences. In addition, a substrate evolves during ubiquitylation, which affects processivity as revealed by recent single-molecule experiments. In a process called processive affinity amplification, ubiquitylation increases the duration of a substrate on APC/C and propensity for further ubiquitylation [73]. Processive affinity amplification is determined in part by mobile elements within the APC/C catalytic core, whereby a substrate-linked Ub binds an unprecedented Ub-binding site that is distinct from the UBE2C–Ub binding site on the RING domain of APC11 [74]. The RING domain simultaneously captures a Ub linked to a substrate and places UBE2C to ubiquitylate another site on a substrate [50]. Accordingly, mutationally eliminating the secondary RING–ubiquitin interaction decreases processivity in vitro, reducing the number of Ubs received by a given substrate molecule, while increasing the number of substrates receiving at least one Ub [50], although other Ub-binding sites within APC/C that contribute to processive affinity amplification or other functions remain to be described.
    APC/C Uses a Dynamic Cullin–RING Mechanism to Elongate Polyubiquitin Chains Human APC/C generates Lys11-linked poly-Ub chains through an entirely different mechanism, via the distinctive E2 enzyme, UBE2S 30, 31, 32. Although APC2 and APC11 are necessary and sufficient to activate UBE2S, the mechanism was not overtly obvious because UBE2S lacks the hallmark E2 residues known to engage RING domains [31]. Indeed, mutation of the APC11 residues binding UBE2C do not impair UBE2S-mediated polyubiquitylation 74, 75. Furthermore, unlike the reaction with UBE2C that is stimulated by coactivator, the intrinsic Ub chain building activity of UBE2S (i.e., transfer of a donor Ub onto a free acceptor Ub) is activated by recombinant APC/C that can be prepared independently of coactivator 21, 74. Instead, a primary function of APC/C is recruiting and positioning the acceptor Ub for its Lys11 to accept another Ub from UBE2S 74, 75. A structural model for the unprecedented interactions between UBE2S, its target (i.e., a Ub that has been linked already to an APC/C substrate), and APC/C was generated by hybrid structural studies merging information from NMR, crosslinking, and mutational data with a low-resolution cryo-EM map of APC/CCDH1 complexed with a proxy for the Ub chain elongation intermediate (Figure 4C) [50]. Although the details of these interactions await high-resolution studies, it is clear that APC/C engages UBE2S in a bipartite manner, but this differs completely from interactions with UBE2C. UBE2S is anchored to APC/C by a flexibly tethered extension C terminus of the catalytic domain of UBE2S 30, 31, 32. Here, the extreme C-terminal residues of UBE2S pack into a pocket between the APC2 N-terminal domain and APC4 β-propeller 47, 50. Additionally, the UBE2S catalytic domain interacts with an APC2 surface that differs completely from previously described E3–E2 interactions 50, 74, 75. The RING is also crucial, as it possesses a distinctive binding site recruiting Ub for modification by UBE2S [74]. Apparently, the catalytic geometry whereby the RING domain of APC11 can present Ub for modification by UBE2S is attainable even with APC2-APC11 in the “down position”, which accounts for APC/C activating UBE2S-dependent generation of unanchored poly-Ub chains even in the absence of a coactivator and a substrate 21, 52, 74, 75. Overall, the data suggest that UBE2S can be activated by various positions of the APC2–APC11 cullin–RING catalytic core, although at this point, it remains unknown whether the position of the APC11 RING domain required to engage UBE2C allows simultaneous positioning of Ub for modification by UBE2S, or how UBE2C and UBE2S might cofunction with APC/C to generate branched Ub chains.