RING-between-RINGs--keeping the safety on loaded guns.

The ‘RING-between-RING’-type E3 ubiquitin ligase HOIP acts via a novel RING/HECT-hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non-covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is autoinhibited. This explains why assembly of either HOIP/ HOIL-1L or HOIP/SHARPIN is required to catalyse linear chain formation. Post-translational modification of a protein with Ubiquitin (Ub) requires the activity of three enzymes: a Ub activating enzyme (E1), a Ub conjugating enzyme (E2), and a Ub ligase (E3). Final Ub transfer is performed by an E3 enzyme, which mediates the ligation of Ub from an E2BUb conjugate (‘B’ denotes a thioester) onto a substrate. E3s are commonly divided into two mechanistic classes: RING/U-box E3s and HECT E3s. RING/U-box E3s facilitate the transfer of Ub from the E2BUb directly onto a substrate amino group. In contrast, HECTs transfer Ub from the E2BUb to the substrate via a HECTBUb intermediate. This mechanistic difference leads to an important distinction regarding what determines the type of Ub product (i.e., the specific Ub-chain linkage) formed: in ubiquitination pathways involving RINGtype E3 ligases, the E2 determines the product formed, whereas for HECT-catalysed pathways, the E3 governs product formation (Christensen et al, 2007; Kim and Huibregtse, 2009). RING-between-RING (RBR) E3s comprise a class of E3s that appear to have special properties. Although RBR E3s have been considered as a subfamily of RING E3s, the RBR E3 HHARI (Human Homologue of ARIadne) was recently shown to form a HECT-like E3BUb intermediate (Wenzel et al, 2011). Two other members of the RBR family, HOIL-1 and HOIP, form the Linear Ub Chain Assembly Complex (LUBAC), the only E3 ligase known to catalyse the synthesis of linear Ub chains (Kirisako et al, 2006). Linear Ub chains are produced by head-to-tail conjugation of Ub molecules through their Nand C-termini and have been shown to activate the canonical NF-kB pathway (Tokunaga et al, 2009). Two studies by the Rittinger and Sixma groups now reveal important insights regarding the formation of linear Ub chains by the dimeric RBR E3 complex HOIP/HOIL-1L (Smit et al, 2012; Stieglitz et al, 2012). Results from these studies highlight three emerging themes among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions. The RBR E3 ligase domain consists of two distinct RING domains, called RING1 and RING2, connected by an IBR (In-Between-Ring) domain. Despite its name, RING2 is not a canonical RING domain as it contains an active site Cysteine (Cys), which has recently been shown to form a thioester E3BUb intermediate, as directly detected for the RBR E3 HHARI. Although the Ub-loaded species could not be detected for the RBR E3 parkin, mutation of the analogous cysteine residue abrogated parkin’s ligase activity implying that it works via the same mechanism. On the basis of these observations, Wenzel et al (2011) proposed that the RBR E3s are a family of RING/HECT hybrids that use RING1 to bind an E2 (RING-like) and RING2 to present the active site Cys (HECT-like) as shown schematically in Figure 1. Both Smit et al (2012) and Stieglitz et al (2012) observed a HOIPBUb thioester, confirming that HOIP also acts via a RING/ HECT-hybrid mechanism. Furthermore, Smit et al (2012) used a clever strategy to uncouple the first transfer event