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  • Here we describe preparation of three stable conjugates

    2021-02-27

    Here, we describe preparation of three stable conjugates that are linked by either oxyester, disulfide, or isopeptide bonds (Fig. 10.1). Each of these conjugates depends upon the prior purification of E2 and ubiquitin proteins that have been engineered to favor specific linkages. For the oxyester- and isopeptide-linked conjugates wild-type ubiquitin is used, and it is only necessary to mutate the active site Cys of the E2 to either Ser or Lys, respectively (Fig. 10.1). However, formation of the disulfide-linked conjugate requires EPI-001 of the C-terminal Gly in ubiquitin to Cys. Some E2s, such as UBE2B, only possess one Cys at the catalytic site, and the wild-type protein can be used for conjugation. Whereas others, such as UbcH5b, have several additional Cys that must be mutated to Ser to avoid formation of cross-linked E2s, rather than the desired E2~ubiquitin disulfide. Formation of the disulfide-linked conjugate is not dependent on E1. However, active E1 is required for the preparation of both the oxyester- and isopeptide-linked conjugates. In a number of E2s, the UBC domain contains an additional noncatalytic “backside” ubiquitin-binding site (Brzovic et al., 2006, Sakata et al., 2010). Interaction of ubiquitin with this site can result in the noncovalent association of conjugate molecules, which may promote chain formation and complicate some analyses. Therefore, mutations are often introduced to disrupt this interaction, and in the case of the widely studied E2, UbcH5b, replacement of Ser 22 with Arg (S22R) is sufficient to disrupt ubiquitin binding to the backside site.
    The structures of several conjugates have been determined. These show that, in the absence of any binding partners, ubiquitin can occupy distinct positions relative to the E2; and depending on the orientation of ubiquitin, the conjugate is referred to as adopting either an “open” or a “closed” conformation (Fig. 10.3; Wenzel, Stoll, & Klevit, 2010). The first structural model of an isolated E2~Ub conjugate was obtained of a thioester-linked conjugate (Hamilton et al., 2001). For these heroic studies, the NMR data were acquired using a sample that contained E1 and ATP, thereby allowing the unstable E2~ubiquitin thioester to be maintained, while the NMR data were collected. This showed that yeast Ubc1 (the yeast homologue of UBE2K) could adopt a closed conformation, whereby the Ile 44 (I44) centered face of ubiquitin interacts with the E2 adjacent to the active site Cys (Fig. 10.3). 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, Scarce (complex) mRNA 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.