br A brief history of
A brief history of RBR E3s RBR E3s were originally defined based on sequence alignments that predicted a tripartite motif of three zinc (Zn)-binding domains: two RING domains (RING1 and RING2) connected via an in-between-RING (IBR) domain , . The prediction together with initial observations of ubiquitination activity led to the belief that RBR E3s constituted a sub-class of the largest E3 class, the RING-type E3s , , , . However, more than 10 years after they were defined, RBR E3s were shown to differ fundamentally from their eponymous RING E3 cousins by virtue of their possessing an active site—a feature lacking in all RING-type E3s . Similar to canonical RINGs, the RING1 domain of the RBR module binds E2s loaded with Ub (E2~Ubs). However, RING2s were found to contain an essential active-site Cys that receives Ub from an E2~Ub to generate a covalent E3~Ub intermediate, otherwise seen only in HECT-type E3s . Based on those two features, RBR E3s are referred to as RING–HECT hybrids. The hybrid mechanism was initially revealed for HHARI and Parkin , but the functional significance of an active-site Cys for catalytic activity has since been confirmed for HOIP, HOIL-1L, TRIAD1, and RNF144A and is now considered a defining feature of RBR E3s , , , . While a useful concept, the description of RBR E3s as RING–HECT hybrids does not fully embrace emerging understanding of their mechanisms. Here, we present current status of the molecular details of how RBR E3s function. Molecular processes that an RBR E3 must undergo during its catalytic AMG 487 clinical include (1) E2~Ub binding, (2) E3~Ub generation, (3) substrate binding, and (4) transfer of Ub to the substrate. While these steps must follow a logical sequence, the precise order of events is still unknown. Furthermore, early biochemical studies of Parkin uncovered a mode of auto-inhibition . Subsequently, several other human RBR E3s were shown to be auto-inhibited, though through different mechanisms , , , , . Each RBR E3 has its own set of protein targets, and each RBR E3 dictates the type of modification (mono-, linear, or poly-Ub chains) that it generates on its targets (Fig. 1). As is the case for the other classes of E3s, identification of substrates is challenging and under-developed. At present, it is not even clear where a protein substrate binds on an RBR E3. How an RBR E3 determines the type of modification is known for only one RBR E3, HOIP (discussed below ) and remains to be defined for all others. As presented in subsequent sections of this article, more progress has been made to define the structural and biochemical underpinnings of steps in the catalytic cycle prior to the ultimate attachment of Ub to substrate.
RBR E3s control the type of product generated That RBR E3s contain an active site distinguishes them from canonical RING-type E3s. This distinction is more than a technicality because it is the enzyme that carries out the final Ub transfer reaction that determines the type of Ub modification (i.e., mono- versus poly-ubiquitination and chain linkage configuration) on a given substrate. In chemical terms, the final step is the only one that is not a transthiolation reaction (i.e., transfer from one Cys to another). Thus, the E2 determines the product type when working with RING-type E3s by transferring the Ub from its active-site Cys to a non-Cys (most often, Lys) residue on the substrate. As a result, RING-type E3s can modify their substrates with different Ub products depending on which E2 it pairs with. However, in the case of E3s that form an E3~Ub intermediate (i.e., HECT- and RBR E3s), the E3 dictates the final Ub product. A clear example of this switching of control between E2 and E3 enzymes is presented in a study of LUBAC (which contains HOIP) with the human E2 known as E2‐25K (Ube2K), that possesses intrinsic ability to build K48-linked Ub chains in the absence of an E3. This chain linkage preference is suppressed when Ube2K works with LUBAC where the E2/E3 pair robustly generates linear Ub chains . In this case, the E2~Ub simply acts as a supplier of Ub to the E3, and must not modify substrates (Lys) directly so as to allow linear chain-building specificity to be conferred by the E3 HOIP. Consequently, HOIP (and presumably other RBR E3s) dictates final product formation on substrates independent of the E2 involved , , .