How do the structurally similar canonical

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 Fosaprepitant dimeglumine salt 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. A structural explanation for how RBR RING1s handle their bound E2~Ubs differently from canonical RINGs has not been readily apparent. Only a few residue positions are strongly conserved in RING and RING1 domains, most of which are zinc-coordinating Cys and histidine residues. A key position in canonical RINGs is the linchpin residue, which is largely responsible for the ability to promote closed E2~Ubs by forming hydrogen bonds to both E2 and Ub [71]. RBRs do not have a conserved residue that can fulfill the linchpin function. This provides a possible explanation for RING1\'s failure to induce closed states of E2~Ub, but it does not explain how a RING1 domain actively promotes open E2~Ubs. In two recent crystal structures of HOIP/UbcH5~Ub and of HHARI/UbcH7~Ub complexes, the E2~Ub bound to the RING1 domain is in an open conformation [47], [48]. When not bound to an E3, UbcH5~Ub is predominantly in open states that exhibit limited aminolysis reactivity [67]. Paradoxically, although the human E2 UbcH7 solely performs transthiolation reactions [34], unbound UbcH7~Ub exists mainly in closed conformations [54], indicating that an as yet unidentified feature of the UbcH7 active site must be responsible for its restricted chemical reactivity profile. The observation of an open UbcH7~Ub bound to HHARI implies that binding to HHARI RING1 either actively favors open states or disfavors closed states. No contacts with any HHARI domains are observed for the Ub moiety of UbcH7~Ub in the HHARI/UbcH7~Ub complex, suggesting that the open state is not stabilized by additional E3 contacts (Fig. 4A) [48], [54]. Instead, an extension of the second Zn-loop of HHARI RING1 is largely responsible for disfavoring the closed E2~Ub conjugate [48]. In canonical RING domains, the final two Zn-coordinating Cys residues are consistently separated by exactly two residues (C7th-X-X-C8th), but the same loop contains up to four residues in RING1s (C7th-X-X-X-X-C8th) [51]. Deletion of the extra residues in the HHARI Zn-loop II to create a two-residue loop generates a RING1 with diminished ability to disrupt closed UbcH7~Ub [48]. It is the loop length rather than a specific side chain that leads to the opening activity, consistent with the lack of conservation in four-residue loops among RBR RING1s. Thus, in the case of HHARI and other RBR E3s with extended Zn-loops, the open E2~Ub conformation is achieved mainly through use of a steric wedge that restricts the conjugate from adopting closed conformations [48].