Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 sidechain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.
Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes including protein quality control, DNA repair, endocytosis and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the Ub C-terminus and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly-linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent “backside” interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the “infinite spiral” helical arrangement of the sole previously reported structure of an oligomeric conjugate. Our structure also differs in intra-conjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intra-conjugate relative E2/Ub orientations. We delineate a common intra-conjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 ligase CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3 ligases, which have important consequences for the ubiquitination process.
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.
transcription; mediator complex; Gcn4; Gal11/Med15; acidic activator; activation domain; intrinsically disordered; NMR
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.
ubiquitin; ubiquitin conjugating enzyme; ubiquitination; UbcH5; Ubc13; NMR; spin label; SAXS
Ubiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field.
Ubiquitination is a post-translational modification pathway involved in myriad cellular regulation and disease pathways. The ubiquitin (Ub) transfer cascade requires three enzyme activities: a Ub-activating (E1) enzyme, a Ub-conjugating (E2) enzyme, and a Ub ligase (E3). Because the E2 is responsible both for E3 selection and substrate modification, E2s function at the heart of the Ub transfer pathway and are responsible for much of the diversity of Ub cellular signaling. There are currently over ninety three-dimensional structures of E2s, both alone and in complex with protein binding partners, providing a wealth of information regarding how E2s are recognized by a wide variety of proteins. In this review, we describe the prototypical E2/E3 interface and discuss limitations of current methods to identify cognate E2/E3 partners. We present non-canonical E2-protein interactions and highlight the economy of E2s in their ability to facilitate many protein-protein interactions at nearly every surface on their relatively small, compact catalytic domain. Lastly, we compare the structures of conjugated E2~Ub species, their unique protein interactions, and the mechanistic insights provided by species that are poised to transfer Ub.
ubiquitin; ubiquitin-conjugating enzyme; ubiquitin ligase enzyme; ubiquitination; ubiquitylation
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.
GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, non-catalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, -5, -6, 10, and -11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, -5, -6, and -10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.
A ubiquitin ligase (E3) functions at the crossroad between ubiquitin activation and the attachment of ubiquitin to protein substrates. During this process, the E3 interacts with both a substrate and a ubiquitin-conjugating enzyme (E2). Although a major goal when investigating an E3 is to identify its substrates, recent evidence indicates that the E2 dictates the type of ubiquitin modification that will occur on the substrate. There are ~ 30 E2s identified in the human genome, many of which remain to be characterized. We found that the RING E3 BRCA1/BARD1 can interact with 10 different E2s. The ability of BRCA1 to interact with multiple E2s is likely to be a common feature among other RING and U-box E3s. We and others have also found that certain E2s show a preference for attaching either the first ubiquitin to a substrate lysine or ubiquitin to itself (chain building), suggesting that E2s may play a role in dictating product formation. Therefore, when investigating the functions of an E3 it is advisable to identify all E2s that interact with the E3 so that these can be used in E3-dependent substrate-ubiquitination assays. We describe a method used to identify all the E2s that interact with BRCA1. Defining the set of E2s that interact with other RING and U-box E3s will open the door for predictive models and lead to a better understand of substrate ubiquitination.
BRCA1; NMR; protein-protein interactions; RING domain; UbcH5; Ubc13; ubiquitin ligase; ubiquitination; ubiquitin-conjugating enzyme; yeast two-hybrid
The small heat shock protein αB-crystallin (αB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric αB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of ~121° between the planes of the β-sandwich formed by α-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with β4 and β8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with β3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit αB oligomer with tetrahedral symmetry.
Atomic level structural information on αB-Crystallin (αB), a prominent member of the small Heat Shock Protein (sHSP) family has been a challenge to obtain due its polydisperse, oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on ∼ 580 kDa human αB assembled from 175-residue, 20 kDa subunits. An ∼100-residue α-crystallin domain is common to all sHSPs and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and the Cβ resonances have been obtained for residues 64-162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid- and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-crystallin domain have similar chemical shifts in both solid- and solution-state indicating a similar structure for the domain in its isolated and oligomeric forms. Sites of inter-subunit interaction are identified from chemical shift differences that cluster to specific regions of the α-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid- and solution-state chemical shift data and (ii) 1H-15N HSQC spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer-monomer transition over the pH range of 7.5 to 6.8. This steep pH-dependent switch may be important for αB to function optimally, e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia which is accompanied by acidosis.
αB-Crystallin; chaperone; sHSP; solid-state NMR; solution-state NMR
The PhoQ sensor-kinase is essential for Salmonella typhimurium virulence for animals, and a homolog exists in the environmental organism and opportunistic pathogen Pseudomonas aeruginosa. S. typhimurium PhoQ (ST-PhoQ) is repressed by millimolar concentrations of divalent cations and activated by antimicrobial peptides and at acidic pH. ST-PhoQ has a periplasmic PAS domain, a fold commonly employed for ligand binding. However, substrate binding is instead accomplished by an acidic patch in the periplasmic domain that interacts with the inner membrane through divalent cation bridges. The DNA sequence encoding this acidic patch is absent from Pseudomonas phoQ (PA-PhoQ). Here, we demonstrate that PA-PhoQ binds and is repressed by divalent cations, and can functionally complement a S. typhimurium phoQ mutant. Mutational analysis and NMR spectroscopy of the periplasmic domains of ST-PhoQ and PA-PhoQ indicate distinct mechanisms of binding divalent cation. The data are consistent with PA-PhoQ binding metal in a specific ligand-binding pocket. PA-PhoQ was partially activated by acidic pH but not by antimicrobial peptides. S. typhimurium expressing PA-PhoQ protein were attenuated for virulence in a mouse model, suggesting that the ability of Salmonella to sense host environments via antimicrobial peptides and acidic pH is an important contribution to pathogenesis.