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  • Among the Ub interactions observed


    Among the ~Ub interactions observed in the HOIP structure is one that involves a RING2 domain (from the non-cognate HOIP) [47]. A similar binding interaction was defined between HHARI RING2 and the hydrophobic patch on Ub by NMR [54]. HHARI RING2 mutations that ablate the interaction inhibit formation of the E3~Ub intermediate and a similar loss of function occurs when the E2~Ub carries mutations in the hydrophobic surface of the attached Ub [54]. The common feature in HOIP and HHARI suggests that interaction between ~Ub and RING2 plays an integral part in the Ub transfer mechanism of RBRs by recruiting RING2 to the E2~Ub bound to RING1. In addition, Ub contacts on IBR domains of Parkin and HOIP observed in crystal structures have been shown to be important for Ub transfer reactions, although the exact roles these interactions play remain to be defined [47], [50]. Considering the multiple domains that are involved and the large domain movements that must occur to bring the active sites of the E2 and the E3 together, a role for multiple interactions involving Ub is not surprising. Thus, while the multitude of interactions involving ~Ub in the HOIP structure may not reflect a HOIP/E2~Ub complex at a single point in time, it likely represents at least several of the possible configurations of RBR domains relative to a bound E2~Ub along the reaction pathway. Other Phleomycin for Ub have been identified on RBR E3s that involve domains outside the RBR module. These are idiosyncratic to a particular RBR and likely impart special functionality. They are also unlikely to involve a Ub that is conjugated to an E2. For example, the relevant Ub to bind at the HOIP LDD is actually the substrate in the linear chain building reaction (and therefore derives from the distal end of a growing chain). Ub binds to the ubiquitin-associated-like domain (UBA-L) of HHARI [48], but the relevant source of Ub (free, part of an E2~Ub, attached to substrate, etc.) is unknown. The same binding site is used by HHARI to bind to the Ub-like protein Nedd8 that is required for the interaction between HHARI and CRLs [22], [23].
    RBR E3s are auto-inhibited and activated by different mechanisms First observations of an auto-inhibitory mechanism was provided by in vitro biochemical studies of Parkin, followed by HOIP and HOIL-1 [35], [36], [37]. Removal of specific domains enhances ubiquitination activity of these enzymes, indicative of an auto-inhibitory mechanism [35], [36], [37]. Additional biochemical evidence now supports auto-inhibition of HHARI, TRIAD1, and RNF144A, suggesting that auto-inhibition is a common feature among RBR E3s [23], [35], [36], [39], [47]. Therefore, the activity and biochemical study of RBR E3s requires a method to release the auto-inhibition. The diverse and complicated domain architecture of RBR E3s leads to different modes of auto-inhibition and consequently, different mechanisms by which each RBR E3 is activated. Furthermore, Acceptor splicing site has become increasingly clear that activation of RBR E3s can be brought about through the actions and interactions of other proteins, including protein kinases and even other E3 Ub ligases. Initial crystal structures of HHARI and Parkin revealed sources for their auto-inhibition: their active-site Cys residues are at least partially occluded by non-RBR domains and the E2-binding RING1 domain and the active-site Cys-containing RING2 domain are far apart (Fig. 2A) [38], [41]. However, different domains are responsible for occlusion of the active site and the relative dispositions of the RBR domains in the Parkin and HHARI structures are substantially different. Large domain movements must occur in either case to allow Ub transfer from the E2~Ub to the RING2 active-site Cys [45], [73]. Consistent with this notion, the long linkers that connect RING1 to IBR and IBR to RING2 are predominantly disordered in the crystal structures, indicating conformational flexibility. Flexible linkers can allow domains to assume positions that are quite distant (as in the auto-inhibited conformations) as well as proximal to each other (as in presumed activated conformations). Part of the linker that connects the IBR and RING2 is helical in an NMR solution structure of HHARI RING2 and in crystal structures of HOIP RING2–LDD and the helix composes part of the RING2 Ub-binding site in HHARI [40], [46], [47]. The observations suggest that the linker may undergo a disorder-to-order transition as part of the structural rearrangements that must occur for RBRs to become active.