These studies established IAP proteins as
These studies established IAP proteins as dimeric RING E3 ligases, but did not account for the essential role of dimerization. In IAPs and related E3s, such as RNF4 and MDM2, dimerization not only depends on contacts from the core RING domain but also residues N- and C-terminal to the RING domain (Budhidarmo, Nakatani, & Day, 2012). A number of mutagenesis studies suggested that the C-terminal residues were required for ubiquitin transfer, and for MDM2 and RNF4 mutations were identified that disrupted ubiquitin transfer but not RING dimerization (Plechanovova et al., 2011, Uldrijan et al., 2007). Together, these studies suggested that a conserved solvent-exposed C-terminal aromatic residue played an essential role in ubiquitylation. Until recently, the purpose of this aromatic side chain remained uncertain.
In the last few years, a molecular understanding of why RING dimerization and the C-terminal residues are critical for ubiquitin transfer by IAPs has become clearer. This appreciation of RING domain function has depended on the availability of stable E2~Ub conjugates that are suitable for in vitro and biophysical studies. Here, we describe the preparation of several E2~Ub conjugates, and experimental approaches that can be used to uncover RING domain function.
All E2 BRD 7552 share a central UBC domain that includes the catalytic Cys that is charged with ubiquitin by the E1. Once charged, a thioester bond between the side chain of the Cys and the C-terminal carboxylic group of Gly76 in ubiquitin links the two proteins (Fig. 10.1). The thioester bond is relatively unstable making this conjugate difficult to purify in significant quantities. Therefore, to undertake many biochemical and biophysical experiments, it is necessary to prepare long-lived conjugates. 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 mutation 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).