The Inhibitor of APoptosis IAP
The Inhibitor of APoptosis (IAP) proteins were first identified as gene products of baculovirus that inhibited apoptosis of infected insect glycyrrhizin (Clem et al., 1991, Crook et al., 1993). Subsequently, the mammalian IAP family was identified (Rothe et al., 1995, Uren et al., 1996). All members of the IAP family possess at least one baculovirus IAP repeat (BIR) domain, a protein–protein interaction module. Many IAPs also contain a RING domain that confers on them the ability to promote their own ubiquitylation (autoubiquitylation), as well as the ubiquitylation of substrate proteins (Mace et al., 2010, Vaux and Silke, 2005). Ubiquitin transfer by IAPs is central to their ability to modulate signaling pathways. For example, cIAP1 promotes the addition of nondegradative ubiquitin signals on RIPK1, and these are required for assembly of the prosurvival signaling complex (Bertrand et al., 2008). In contrast, small molecule compounds that bind to the BIR domains promote cell death by enhancing cIAP autoubiquitylation and subsequent degradation (Varfolomeev et al., 2007, Vince et al., 2007). As well as modulating cell survival, important roles for IAPs in innate immune signaling have also been established (Vandenabeele & Bertrand, 2012), and the RING domain-mediated E3 ligase activity of XIAP is required for nucleotide-binding and oligomerization domain signaling (Damgaard et al., 2012, Damgaard et al., 2013).
The ligase domain in IAPs resembles other RING domains and, as expected, it has a conserved flat surface that interacts with E2 enzymes (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.