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The structures of enzymes that collectively modify proteins by covalent addition of ubiquitin-like protein moieties have provided significant insights into the regulatory pathways they compose and have highlighted the importance of protein flexibility for the mechanism and regulation of the ubiquitination reaction.
Protein modification by the addition of a small ubiquitin-like protein (Ubl) is a mechanism widely employed in eukaryotic cells to control protein activity after translation (reviewed in ). Ubls constitute a growing family of structurally similar molecules that include ubiquitin (Ub), NEDD8 (neural precursor cell expressed, developmentally downregulated 8), SUMO (small ubiquitin-like modifier), ISG15 (interferon-stimulated gene 15), and ATG8 (autophagy 8), and are reviewed in . Ubls become conjugated to target proteins in a three-enzyme cascade. In the first reaction, an E1 (Ubl-activating) enzyme transfers an adenyl moiety from ATP to the C-terminal glycine residue of the Ubl, thus activating the Ubl for subsequent transfer to an E1 cysteine residue to generate a thioester linked E1-Ubl conjugate. In the next reaction, the Ubl is transferred to an E2 (Ubl-conjugating enzyme) active site cysteine residue, while in the final reaction, an E3 (Ubl ligase) catalyses transfer of the activated Ubl to its protein target (reviewed in [3-5]). In some cases, targets are modified by the addition of only a single Ubl, but in others, signal diversity is increased by the addition of chains of Ubls in which Ubl identity, chain length, and linkage type all contribute to specificity (reviewed in [6,7]).
Like many proteins involved in signalling pathways, E1, E2 and E3 enzymes are characterised by modular architectures in which regulator binding and enzymatic activities are segregated either to different domains within the same protein or, as is the case with many E3s, to different proteins. Recent structures determined by X-ray crystallography have provided detailed snapshots of E1-, E2-, and E3-containing complexes caught at different stages in their catalytic cycles and from these structures important insights into their mechanisms have been gained.
The recent determination of the structure of a complex containing the NEDD8-E1 enzyme APPBP1-UBA3, a catalytically inactive variant of its cognate E2 (Ubc12), two NEDD8 molecules and Mg-ATP  (Figure 1a) elegantly demonstrated how a conformational change in an E1 can generate an affinity switch; only when the E1 is doubly loaded with its cognate Ubl does it acquire high affinity for its cognate E2. In this example, the APPBP1-UBA3 E1, once charged with two NEDD8 molecules at its adenylation (A)- and thioester (T)-active sites, transfers the (covalently bound) T-site NEDD8 to Ubc12 (an E2) and then adopts a conformation that promotes the release of the E2-Ubl conjugate. As the E2-binding sites for E1 and for E3 are mutually exclusive, the structure suggests that the E2 must dissociate from the E1 before it can associate with a cognate E3. Indeed, enzymological studies, at least in some cases, have shown that this is in fact the case .
Subsequent determination of the structure of Uba1, a yeast Ub-E1 non-covalently bound to Ub, has demonstrated that the E1 mechanism, though broadly conserved, is subject to variation in detail  (Figure 1b). A comparison of the structures of the two copies of the Uba1-Ub complex in the crystal asymmetric unit further highlights the different positions that the ubiquitin-fold domain (UFD) of the E1 can adopt relative to the other E1 domains. Notably, in this singly Ub-loaded structure, the location of the UFD is similar to that of the UFD in the doubly NEDD8-loaded APPBP1-UBA3 structure and distinct from that observed in structures of NEDD8  and SUMO  E1 enzymes singly Ubl-loaded at the A site. As discussed in Huang et al. , the reorientation of the UFD that accompanies double-loading of the NEDD8 E1 with NEDD8 is critical to subsequent transfer of the covalently bound T-site NEDD8 to the E2. In addition, the canyon formed between the UFD and the catalytic cysteine domain in the Uba1-Ub structure (Figure 1b) is much wider than that seen in APPBP1-UBA3. Taken together, these and other observations suggest that the conformational changes that accompany transfer of Ub from E1 to E2 will be much less dramatic than those associated with transfer of NEDD8 or SUMO.
As with E1 and E2 enzymes, an overriding theme to emerge from recent structural studies of E3s is the functional importance of conformational flexibility. E3s fall into three major subclasses according to the identity of the domain that binds the covalent E2-Ubl complex. These subclasses are referred to as ‘homologous to the E6-AP carboxyl terminus’ (HECT), ‘really interesting new gene’ (RING)/RING-like, and those E3s that have no resemblance to the HECT- or RING-containing enzymes. Recent structural and biochemical studies have begun to elucidate the mechanistic diversity of this broad enzyme class (reviewed in ), but in all examples, a recurrent theme is the importance of conformational flexibility to allow the E3 complexes to bind and modify different protein substrates.
A particular example, well documented by structural studies, is that of the SCF [Skp1 (S-phase kinase-associated protein 1)-cullin-F box] E3 Ub ligase family (reviewed in ). This E3 contains a RING domain that binds to the cullin subunit. The cullin functions as a scaffold within the complex and also binds to the adaptor protein Skp1 . SCF activity is promoted by cullin neddylation  and inhibited by CAND1 (cullin-associated and neddylation-dissociated 1) binding . Skp1 and the cullin together mediate the interaction between a diverse set of F-box-containing proteins that bring substrates to the E2, and E3 catalytic core. Models for SCF E3/E2 complexes have been constructed by superimposing structures of selected subcomplexes [14,17] (Figure 2a). However, it is difficult to see how catalysis will proceed as the E3 substrate bound to its cognate F-box protein is located approximately 50 Å away from the bound activated E2-Ubl .
The structure of a neddylated cullin-Rbx1 (ring-box 1) complex  (Figure 2b) and enzymological studies of selected SCFs [15,18] together provide valuable extra information to help solve this puzzle. Previous attempts to generate models for an SCF E3 complex had employed structures that contained a non-neddylated cullin subunit. A comparison of the neddylated and non-neddylated cullin structures bound to Rbx1 reveals that neddylation leads to the adoption by the cullin-Rbx1 complex of a more open structure in which the Rbx1 N-terminal linker is no longer constrained by interactions with the cullin, and as a result, the Rbx1 RING domain is also freed so that bound substrate is predicted to come significantly closer to charged E2 . This prediction is supported by the fact that the neddylated SCFβ-TrCP complex, but not the non-neddylated form of this complex, promotes the BMPS [N-(beta-maleimidopropyloxy)succinimide ester]-mediated crosslinking of UbcH5c (an SCFβ-TrCP E2) with a peptide from β-catenin (an SCFβ-TrCP substrate) .
As a further result of these structural changes, the cullin CAND1-binding site is also lost. Thus, the structure of the neddylated cullin-Rbx1 complex answers the question of how the distance between the bound substrate and E2 might be spanned and of how CAND1 inhibitor binding and cullin neddylation reciprocally regulate SCF activity: CAND1 binding stabilises the closed (inactive) cullin structure, whereas cullin neddylation promotes the more open (active) conformation and simultaneously prevents CAND1 association.
Now that initial structures have given a glimpse of the structural diversity of the enzymes of the Ubl conjugation pathway, future work will focus on characterising complete complexes at different stages of their catalytic cycles. We expect further insights into this important signalling mechanism to emerge from visualising and characterising both the conserved and the unique mechanistic aspects of different Ubl-conjugating enzymes.
The authors thank M Noble for his critical reading of the manuscript and the Medical Research Council (UK) for financial support.
The electronic version of this article is the complete one and can be found at: http://F1000.com/Reports/Biology/content/1/19
The authors declare that they have no competing interests.