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  • A structural explanation for how RBR


    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]. GSK461364 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]. The steric wedge model provides a structural explanation for some RBR E3s. But not all family members have four-residue Zn-loops II. Parkin has a three-residue loop and two other RBRs, including HOIP, have two-residue loops. A notable difference between HHARI and Parkin is that auto-inhibited HHARI can bind E2~Ub with high affinity, whereas auto-inhibited Parkin has modest E2 binding at best because its E2-binding site is partly occluded by the inhibitory element known as the repressor element (REP) (Figs. 1A and 2A) [38], [41], [48]. It may therefore be less important for Parkin to actively disfavor reactive closed E2~Ub states than it is for HHARI, as Parkin may only be able to bind an E2~Ub effectively once it is in an activated state. HOIP contains a short two-residue Zn-loop II, yet in the crystal structure in complex with UbcH5~Ub, the conjugate is in an open conformation (Fig. 4B). UbcH5~Ub exists predominantly in open states on its own, so it may not require a specific mechanism to maintain the open state of UbcH5. Even so [and in contrast to HHARI/UbcH7~Ub in which no contacts to the ~Ub moiety (Ub of E2~Ub) are observed] there are non-covalent contacts from the ~Ub of UbcH5~Ub to all three domains in the HOIP RBR that may serve to position the ~Ub moiety within the complex and minimize the chances that a closed E2~Ub will form (Fig. 4B). The multiple contacts observed for a single ~Ub in the HOIP/UbcH5~Ub structure as shown in Fig. 4B involve two chains of HOIP (due to a dimer swap in the crystal, [47]). For example, the Ub moiety of UbcH5~Ub is seen bound to IBR domains from two different HOIP polypeptides. Reported evidence for a HOIP dimer existing in solution is equivocal so it is not clear if the observed interactions occur simultaneously within a HOIP/UbcH5~Ub complex [47]. In this complex, the structure of a single HOIP polypeptide may still reflect a less active state, as the RING2 active site is far from the active site of the bound E2~Ub (Fig. 4B). Despite that caveat, it is likely that some, if not all, of the observed contacts involving Ub take place at some point along the reaction pathway, although the details remain to be fleshed out.