SKF 81297 hydrobromide australia The ligase domain in IAPs r

The ligase domain in IAPs resembles other RING domains and, as expected, it has a conserved flat surface that interacts with E2 SKF 81297 hydrobromide australia (Mace et al., 2008). Ubiquitin transfer by IAPs depends on both the integrity of the E2-binding site and RING dimerization (Feltham et al., 2011). Disruption of the dimer abrogates activity and although the isolated RING domain from all IAP proteins forms a stable dimer, the longer forms of some IAPs, such as cIAP1, are largely monomeric and ubiquitin transfer is impeded. The structure of the autoinhibited monomeric form of cIAP1 showed that the RING dimer interface is occluded due to interactions with the third BIR domain (Dueber et al., 2011). Remarkably, the interaction interface on the BIR domain includes the pocket to which a number of proteins and small molecule compounds bind, and this structure also explained why addition of small molecule BIR-binding compounds promotes RING dimerization and autoubiquitylation of cIAP1 (Dueber et al., 2011). 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 enzymes 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.