In contrast crystal structures of the

In contrast, crystal structures of the oxyester-linked Ubc13 and UbcH5b conjugates (Eddins et al., 2006, Sakata et al., 2010), and the NMR structure of the disulfide-linked UbcH8 conjugate (Serniwka & Shaw, 2009), revealed distinct open conformations (Fig. 10.3). In part, differences in the position of ubiquitin may be due to crystal packing. In the UbcH5b structure, the ubiquitin moiety of one conjugate is bound to the backside surface of another E2 molecule (Sakata et al., 2010). This interaction precludes formation of the closed conformation because ubiquitin contacts the backside of UbcH5b with the same surface that contacts E2 in the closed Ubc1~Ub structure. It is also possible that the favored orientation of ubiquitin differs for each E2. Comparison of the dynamic properties of Ubc13 and UbcH5b conjugates supports this hypothesis, as Ubc13 adopts the closed conformation more frequently than UbcH5c (Pruneda, Stoll, Bolton, Brzovic, & Klevit, 2011). With various forms of the E2~ubiquitin conjugates in hand, as well as an initial understanding of the dynamic properties of these molecules, it DNA Damage DNA Repair Library is possible to characterize ubiquitin transfer by RING E3 ligases. Current studies focus on understanding how RING domains promote ubiquitin transfer, and formation of polyubiquitin chains. Here, we describe techniques routinely used in our laboratory to study RING domains from IAP proteins, although the approaches described could be readily applied to other E3s.
Conclusion In the last year, significant advances in our understanding of ubiquitin transfer by IAPs and related E3 ligases have been made. The three crystal structures of the RING-bound E2~Ub show that the conjugate is bound by the RING domain in the closed conformation, where the I44-centered face of ubiquitin makes extensive contacts with the E2 (Dou et al., 2012, Dou et al., 2013, Plechanovova et al., 2012). This conformation is stabilized by RING–ubiquitin interactions; either an aromatic residue on the C terminus of the dimerized RING, or a region N-terminal to the ligase domain. These interactions place ubiquitin in a conformation that is thought to prime the thioester bond for nucleophilic attack by a substrate Lys/N-terminal Met. This model accounts for much of the prior biochemical data, and explains why RING dimerization and an aromatic residue at the dimer interface are required for ubiquitin transfer of IAPs (Dou et al., 2012, Nakatani et al., 2013). Despite advances in our understanding of the overall topology of the RING-E2~Ub complex (Dou et al., 2012, Dou et al., 2013, Plechanovova et al., 2012), and the importance of reducing conformational freedom for catalysis (Berndsen et al., 2013), a detailed mechanistic understanding of ubiquitin transfer from the E2 to a substrate Lys remains uncertain. In addition, although many features of the mode of binding and mechanism of transfer are likely to be conserved in most E2s, the RING-bound structure of a conjugate is only available for highly similar UbcH5 family members. Do other less similar E2~Ub conjugates adopt an analogous configuration when they are bound by their cognate RING domains? Likewise, only a handful of RING proteins have been characterized, and it seems likely that residues outside the core RING domain will be important for stabilization of the closed conformation in some cases (Dou et al., 2013). A molecular understanding of the complexes formed by other E2~Ub conjugates and RING E3s is eagerly awaited.