SUMMARY

Ubiquitylation entails the concerted action of E1, E2 and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C-terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a “fuzzy complex” where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.

Although the functional interaction between ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signaling, the criteria that define an active E2–E3 pair are not well-established. The human E2 UbcH7 (Ube2L3) shows broad specificity for HECT-type E3s1, but often fails to function with RING E3s in vitro despite forming specific complexes2–4. Structural comparisons of inactive UbcH7/RING complexes with active UbcH5/RING complexes reveal no defining differences3,4, highlighting a gap in our understanding of Ub transfer. We show that, unlike many E2s that transfer Ub with RINGs, UbcH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UbcH7 exhibits activity with the RING-In Between-RING (RBR) family of E3s that includes Parkin and human homologue of ariadne (HHARI)5,6. Found in all eukaryotes7, RBRs regulate processes such as translation8 and immune signaling9. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn2+-binding domains, In-Between-RING (IBR) and RING2 domains, which together define this E3 family7. Here we show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ‘~Ub’), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UbcH7, an E2 involved in cell proliferation10 and immune function11, and suggest a novel mechanism for an entire class of E3s.

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for Ub transfer to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub, in solution as determined by NMR and SAXS. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: UbcH5c~Ub populates an array of extended conformations and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces Helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly regarding interaction with Ub ligases.

Substantial evidence has accumulated indicating a significant role for oligomerization in the function of E3 ubiquitin ligases. Among the many characterized E3 ligases, the yeast U-box protein Ufd2 and its mammalian homolog E4B appear to be unique in functioning as monomers. An E4B U-box domain construct (E4BU) has been sub-cloned, over-expressed in E. Coli and purified, which enabled determination of a high resolution NMR solution structure and detailed biophysical analysis. E4BU is a stable monomeric protein that folds into the same structure observed for other structurally characterized U-box domains, all of which are homodimers. Multiple sequence alignment combined with comparative structural analysis reveals substitutions in the sequence that inhibit dimerization. The interaction between E4BU and the E2 conjugating enzyme UbcH5c has been mapped using NMR and this data has been used to generate a structural model for the complex. The E2 binding site is found to be similar to that observed for dimeric U-box and RING domain E3 ligases. Despite the inability to dimerize, E4BU was found to be active in a standard autoubiquitination assay. The structure of E4BU and its ability to function as a monomer are discussed in light of the ubiquitous observation of U-box and RING domain oligomerization.