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  • HOIP s ability to build linear Ub chains


    HOIP's ability to build linear Ub chains arises from a unique domain that follows directly after the RING2 domain, the linear ubiquitin chain-determining domain (LDD) (Fig. 1). Structures have revealed that the LDD is structurally integrated into RING2, creating BI-D1870 mg a single RING2–LDD unit (Fig. 2D) [40], [47]. The LDD binds an acceptor Ub (i.e., the substrate) and orients its N-term toward the RING2 active-site Cys, thereby ensuring linear Ub chain formation in which the C-terminus (C-term) of a donor Ub attached to the E3 active site is covalently linked to the N-term of an acceptor Ub (Fig. 2D) [40]. HOIP is the only RBR E3 that contains an LDD, consistent with the HOIP-containing LUBAC being the only E3 complex known to generate linear Ub chains. HOIP's high specificity for the N-term of the acceptor Ub raises the question of how LUBAC transfers the initial Ub to a substrate Lys. Currently, there are two proposed models: (1) attachment of the initial Ub is facilitated by other LUBAC components [55], [56], and/or (2) LUBAC substrates are first modified (by another E2/E3 pair) with K63-linked poly-Ub chains and these serve as a template onto which HOIP builds linear chains [14], [57]. Product specificities and how these are dictated are much less well defined for other RBR E3s. There is strong biochemical evidence that HHARI is a mono-ubiquitinating E3 [22], [48], but the structural basis for this BI-D1870 mg preference has yet to be uncovered. Parkin is reported to be responsible for a variety of product types that include K6-, K11-, K48-, and K63-linked chains [58], suggesting either that the E3 lacks chain-type specificity or that this is dictated by as yet unknown factors.
    RBR E3 ubiquitin Ub transfer mechanism Insights into the steps of the RBR mechanism that involve the E2~Ub have been forthcoming from recent studies and structures [47], [48], [50], [54]. Several RBR E3s have been shown to be active with more than one E2 [6], [23], [32], [34], [35], [36], [37], [54], [59], [60], [61], but a full description of the cadre of E2s (there are more than 30 in humans) that work with each human RBR E3 is not yet available. Two available structures of RBR E3s in complex with E2~Ub include the two well-characterized human E2s, Ube2D2 (UbcH5b) and Ube2L3 (UbcH7). Importantly, UbcH5 (and most other E2s) can perform both transthiolation reactions and aminolysis reactions, explaining how it can be active with either RBR E3s or RING-type E3s [34], [35], [36], [59], [60], [61]. The human E2 UbcH7 is a more specialized enzyme that solely performs transthiolation reactions [34], implying that it can function with HECT- and RBR-type E3s, but not with RING-type E3s. Paradoxically, UbcH7 can bind to some canonical RINGs [62], [63], but is not active for Ub transfer with them. It is not yet known whether UbcH7 is a biologically relevant E2 for all RBR E3s, but a strong case can be made for HHARI and HOIP. The orthologs of HHARI and UbcH7 act together in a critical developmental step of the gastro-intestinal system in C. elegans[17], [64]. UbcH7 (but not UbcH5) is able to activate NF-κB reporters in cell culture, and some alleles of UbcH7 have been linked to auto-immune diseases suggesting that UbcH7 is a biologically relevant E2 for HOIP in vivo[14], [65], [66]. How do the structurally similar canonical RINGs and RBR RING1s (Fig. 2B) direct a bound E2~Ub to perform different reactions—aminolysis in the case of RINGs and transthiolation in the case of RING1s? On their own, E2~Ub species are highly dynamic and flexible [67]. Upon binding to canonical RING domains, E2~Ubs adopt closed states in which the hydrophobic surface of Ub is buried in contact with the E2 (Fig. 3A) [68], [69], [70], [71], [72]. As closed E2~Ub states display increased reactivity toward Lys amino groups [71], canonical RINGs facilitate the direct transfer of Ub from its bound E2~Ub to substrate. In contrast, RBR RING1 domains of HHARI and RNF144A actively disfavor closed E2~Ubs (Fig. 3B) [54]. By promoting open E2~Ub, RBRs ensure that Ub transfer occurs through their E3 active site by reducing E2~Ub reactivity toward Lys residues (which would be off-pathway, [54]). The open E2~Ub conformation also reveals the hydrophobic patch of Ub that is otherwise buried in the E2/Ub interface in closed E2~Ub conformations (Fig. 3) [54]. Both ramifications of the open E2~Ub are mechanistically important, as discussed below.