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  • In all available E E structures the RING type

    2020-11-13

    In all available E2:E3 structures, the RING-type domain binds the E2 on a surface that is remote from the active site Cys (and therefore from the ubiquitin thioester) (Fig. 2). The non-contiguous E3-binding and active sites on the E2 imply that the role played by a RING to facilitate ubiquitin transfer may be indirect and, therefore, allosteric. However, apo- and E3-bound E2 structures are largely indistinguishable and fail to suggest a mechanism for the allostery. Recent structural studies characterizing the interactions of the more relevant E2~Ub conjugated species with RING-type domains have provided much needed insight. Notably, a solution-based study of E2~Ub conjugates established their dynamic nature and that E2 and the thioester-linked ubiquitin adopt an array of ‘open’ and ‘closed’ conformations [91] (Fig. 4A). Three structures of E2~Ub conjugates of the UbcH5 family (Ube2D1-3) in complex with RING-type E3s (RNF4:UbcH5A~Ub, E4B:UbcH5C~Ub, and BIRC7:UbcH5B~Ub) provide the first glimpses at an E3:E2 complex poised to transfer ubiquitin [13], [92], [93]. A striking common feature is a ‘closed’ conformation of the E2~Ub conjugate in which the Ile44 Wiskostatin surface of ubiquitin is positioned against the 310 helix, active site, and helix 2 of E2 (Fig. 4A–C). In solution, where multiple species can exist simultaneously, E3 binding promotes a population shift in the highly flexible E2~Ub towards closed conformations which, based on activity data, primes the active site for transfer (Fig. 4A) [13], [93], [94]. The closed E2~Ub states are readily disrupted and even conservative mutations of hydrophobic residues in the interface between E2 and ubiquitin can destabilize the closed state and greatly decrease E3-stimulated ubiquitin transfer [93]. It is interesting to note that E2~Ub conjugates that populate closed conformations to a significant extent in the absence of an E3, such as Ubc13 [91], Ubc1 [95], Ube2S [96], and Cdc34 [97], also demonstrate E3-independent ubiquitin transfer. In contrast, the UbcH5 family of E2s, which shows robust activity with a large number of RING-type E3s, has almost undetectable E3-independent activity, consistent with its populating mainly open states in the absence of an E3 [91]. Several non-mutually exclusive possibilities for how RING binding promotes closed E2~Ub conformations are suggested by the recent RING-type E3:E2~Ub structures. One possibility is through direct RING–ubiquitin interactions. Available structures, however, show that the extent to which the RING directly interacts with ubiquitin varies among E3s (Fig. 4B and C). The interleaved homodimeric RING structures of BIRC7 and RNF4 show additional interactions required for activity between the opposing (i.e. non-E2-binding) RING and the conjugated ubiquitin [13], [92], [98] (Fig. 4C, see the C-terminus of BIRC7 highlighted in red). As discussed above, E3s including Mdm2–MdmX, XIAP, and IDOL also adopt an interleaved RING dimer structure and therefore may adopt a similar strategy to enhance closed E2~Ub states. However, monomeric RING-type E3s such as the U-box E3 E4B, or dimeric E3s such as BRCA1–BARD1, which dimerize through regions outside of their RINGs, either lack the additional ubiquitin-interacting surface used by the interleaved dimers or this surface is not available to interact with ubiquitin. The presence or absence of additional RING contacts with ubiquitin is consistent with reports that some E3s exhibit a higher affinity towards E2~Ub than the isolated E2, while others bind them with affinities that are indistinguishable. A second non-conflicting possibility for how RING binding promotes closed E2~Ub conformations is through allosteric activation in the context of the E2~Ub. All three of the recent RING-type E3:E2~Ub structures contain an Arg in loop 2 of the RING-type domain that is critical for E3-enhanced activity (e.g.Fig. 4D, Arg286 of BIRC7, shown in blue). Crystal structures of the dimeric RNF4 and BIRC7 complexes show the side chain of this Arg hydrogen bonding with the backbone of the E2 (Gln92), the backbone of ubiquitin (Arg72), and the side chain of ubiquitin residue Gln40 (Fig. 4D). While mutation of the analogous Arg (Arg1143Ala) in the monomeric E4B only modestly decreases its binding affinity for E2, it leads to a substantial loss of closed E2~Ub conformations. Coincident with this is a disappearance of select NMR spectral perturbations near the E2 310 helix associated Wiskostatin with functional E2 binding and the formation of closed E2~Ub conformations. The Arg-induced alterations near the E2 310 helix span much of the distance between the RING-binding surface and active site of E2, prompting Pruneda et al. to propose that the interaction between this Arg and the backbone of E2 is the allosteric ‘link’ underlying the observed E2~Ub closed conformation and rearrangements near the active site (Fig. 4D, magenta). Notably, a similar allosteric path had been proposed previously for UbcH5 on the basis of a statistical coupling analysis of E2 primary sequences [99]. Interestingly, as noted above, the analogous RING loop 2 Arg also contacts ubiquitin in the crystal structures of RNF4 and BIRC7, and mutation of ubiquitin Gln40 (Gln40Ala or Gln40Arg) affects E3-enhanced activity. However, the significance of the Arg interaction with Gln40 in promoting E2~Ub closed confirmations in the context of RING binding awaits further analysis. Notably, only an Arg at this position can provide multiple hydrogen-bond donors; even a Lys, the next the most prevalent amino acid found at this position in RING-type domains, cannot do so.