IAPs: Modular regulators of cell signalling.

Members of the inhibitor of apoptosis (IAP) family are characterised by the presence of at least one baculoviral IAP repeat (BIR) domain. However, during the course of evolution, other globular modules have been adopted to perform distinct functions. Consequently, the IAP family is now recognised as consisting of members that perform critical functions in different aspects of cellular regulation. In this review, the structural diversity present within the IAP protein family is presented. Known structures of individual domains are discussed and their properties are described in light of recent data. In particular the plasticity of BIR domains and their ability to accommodate different binding partners is highlighted, as well as the importance of communication between the domains in regulating the covalent attachment of ubiquitin.

RINGs hold the key to ubiquitin transfer.

Ubiquitylation, the covalent modification of proteins by the addition of ubiquitin, relies on a cascade of enzymes that culminates in an E3 ligase that promotes the transfer of ubiquitin from an E2 enzyme to the target protein. The most prevalent E3 ligases contain a type of zinc-finger domain called RING, and although an essential role for the RING domain in ubiquitin transfer is widely accepted, the molecular mechanism by which this is achieved remains uncertain. In this review, we highlight recent studies that have suggested that the RING domain modulates the stability of the E2-ubiquitin conjugate so that catalysis is promoted. We also review the role of RING dimerisation and emphasise the importance of studying RING domains in the context of the full-length protein.

The ubiquitin-associated domain of cellular inhibitor of apoptosis proteins facilitates ubiquitylation.

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases.

Smac mimetics activate the E3 ligase activity of cIAP1 protein by promoting RING domain dimerization.

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. IAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer. However, addition of either mono- or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways.

The RING domain of RING Finger 11 (RNF11) protein binds Ubc13 and inhibits formation of polyubiquitin chains.

The Really Interesting New Gene (RING) Finger protein 11 (RNF11) is a subunit of the A20 ubiquitin-editing complex that ensures the transient nature of inflammatory responses. Although the role of RNF11 as a negative regulator of NF-κB signalling is well-documented, the molecular mechanisms that underpin this function are poorly understood. Here, we show that RNF11 binds both Ubc13 and the Ubc13~ubiquitin conjugate tightly and with similar affinity, but has minimal E3 ligase activity. Remarkably, RNF11 appears to bind Ubc13 so tightly that it outcompetes the E1 and an active E3 ligase. As a consequence, RNF11 may regulate the activity of E3s that rely on Ubc13 for ubiquitin chain assembly by limiting the availability of Ubc13 and its conjugate.

The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1.

Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains. Here, we show that TRAF6 RING dimers form a catalytic complex where one RING interacts with a Ubc13~Ubiquitin conjugate, while the zinc finger 1 (ZF1) domain and linker-helix of the opposing monomer contact ubiquitin. The RING dimer interface is conserved across TRAFs and we also show that TRAF5-TRAF6 heterodimers form. Importantly, TRAF5 can provide ZF1, enabling ubiquitin transfer from a TRAF6-bound Ubc13 conjugate. Our study explains the dependence of activity on TRAF RING dimers, and suggests that both homo- and heterodimers mediated by TRAF RING domains have the capacity to synthesise ubiquitin chains.

Use of E2~ubiquitin conjugates for the characterization of ubiquitin transfer by RING E3 ligases such as the inhibitor of apoptosis proteins.

Ubiquitylation of proteins is a versatile posttranslational modification that can serve to promote protein degradation, or it can have nondegradative roles, such as mediating protein-protein interactions. The Inhibitor of APoptosis (IAP) proteins are important regulators of pathways that control cell death, proliferation, and differentiation. A number of IAP family members are RING E3 ubiquitin-protein ligases, which promote direct transfer of ubiquitin from charged E2 enzymes, or E2~ubiquitin (E2~Ub) conjugates, to substrate proteins. This results in the attachment of nondegradative ubiquitin signals to other proteins, or the autoubiquitylation and degradation of IAPs. Modulating ubiquitin transfer by IAPs is the focus of a number of drug development initiatives and these studies require a detailed understanding of ubiquitylation. Here, we describe preparation of stable E2~Ub conjugates that can be used in biochemical and biophysical experiments to examine RING domain function. In the last 2 years, the availability of these conjugates has helped unveil a molecular understanding of the process of ubiquitin transfer by IAPs. The approaches described here will be suitable for studying other RING E3 ligases.