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. Keywords ubiquitin; ubiquitin conjugating enzyme; ubiquitination; UbcH5; Ubc13; NMR; spin label; SAXS Covalent attachment of the 8.6 kDa Ubiquitin (Ub) to target proteins is an essential step in eukaryotic signaling pathways. The type of Ub modification can vary, inducing distinct signals. For example, mono-ubiquitination may elicit a signal for protein transport while poly-ubiquitination (attachment of a chain of Ubs to the target) may mark a protein for proteasome-mediated degradation. Covalent attachment of Ub to a substrate proceeds through a multi-enzyme process consisting of a Ub-activating enzyme (E1), a Ubconjugating enzyme (E2), and a Ub ligase (E3) (1). The human genome contains two Ub E1s, ~35 E2s, and many hundreds of E3s (2). Despite the large number of E2s, they share significant similarity at both the sequence and structure levels. The E2 plays a central role in the ubiquitination cascade, shuttling Ub from an E1 to an E3/substrate complex. The E2 (6), and 3JW0 (7)). In each case, the backbone of the E2 and Ub moieties are not significantly altered from their free structures. However, each structure presents a unique relative orientation of the E2 and Ub units.Despite the wealth of atomic-level structural information on E2s, on E2/E3 complexes, and a growing number of E2~Ub conjugates, the ways in which E3s catalyze Ub transfer from an E2~Ub remain poorly understood. It is clear that one function of the E3 is to bind a substrate simultaneously with binding an E2~Ub, thereby bringing the two components into proximity. However, even in the absence of a protein substrate, E3s have been shown to enhance the rate at which Ub is released ...

Synopsis 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.

The streptococcal coaggregation regulator (ScaR) of Streptococcus gordonii is a manganese-dependent transcriptional regulator. When intracellular manganese concentrations become elevated, ScaR represses transcription of the scaCBA operon, which encodes a manganese uptake transporter. A member of the DtxR/MntR family of metalloregulators, ScaR shares sequence similarity with other family members, and many metal-binding residues are conserved. Here, we show that ScaR is an active dimer, with two dimers binding the 46-bp scaC operator. Each ScaR subunit binds two manganese ions, and the protein is activated by a variety of other metal ions, including Cd2+, Co2+ and Ni2+, but not Zn2+. The crystal structure of apo-ScaR reveals a tertiary and quaternary structure similar to its homolog, the iron-responsive regulator DtxR. While each DtxR subunit binds a metal ion in two sites, labeled primary and ancillary, crystal structures of ScaR determined in the presence of Cd2+ and Zn2+ show only a single occupied metal binding site that is novel to ScaR. The site analogous to the primary site in DtxR is unoccupied, and the ancillary site is absent from ScaR. Instead, metal ions bind to ScaR at a site labeled “secondary”, which is composed of Glu80, Cys123, His125 and Asp160 and lies roughly 5 Å away from where the ancillary site would be predicted to exist. This difference suggests that ScaR and its closely related homologs are activated by a mechanism distinct from that of either DtxR or MntR.

Protein-engineering methods (Φ-values) were used to investigate the folding transition state of a lysin motif (LysM) domain from Escherichia coli membrane-bound lytic murein transglycosylase D. This domain consists of just 48 structured residues in a symmetrical βααβ arrangement and is the smallest αβ protein yet investigated using these methods. An extensive mutational analysis revealed a highly robust folding pathway with no detectable transition state plasticity, indicating that LysM is an example of an ideal two-state folder. The pattern of Φ-values denotes a highly polarised transition state, with significant formation of the helices but no structure within the β-sheet. Remarkably, this transition state remains polarised after circularisation of the domain, and exhibits an identical Φ-value pattern; however, the interactions within the transition state are uniformly weaker in the circular variant. This observation is supported by results from an Eyring analysis of the folding rates of the two proteins. We propose that the folding pathway of LysM is dominated by enthalpic rather than entropic considerations, and suggest that the lower entropy cost of formation of the circular transition state is balanced, to some extent, by the lower enthalpy of contacts within this structure.

The ubiquitin (Ub)-conjugating enzymes Ubc4 and Ubc5 are involved in a variety of ubiquitination pathways in yeast, including Rsp5-and anaphase-promoting complex (APC)-mediated pathways. We have found the double deletion of UBC4 and UBC5 genes in yeast to be lethal. To investigate the essential pathway disrupted by the ubc4/ubc5 deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation C86A and the E3-binding mutations A97D and F63A were both unable to rescue the lethal phenotype, indicating that an active E3/E2ϳUb complex is required for the essential function of Ubc4/Ubc5. A mutation that specifically eliminates RING E3-catalyzed isopeptide formation but not HECT E3 transthiolation (N78S-Ubc4) rescued the lethal phenotype. Thus, the essential redundant function performed by Ubc4 and Ubc5 in yeast is with a HECT-type E3, likely the only essential HECT in yeast, Rsp5. Our results also suggest that Ubc1 can weakly replace Ubc4 to transfer mono-Ub with APC, but Ubc4 cannot replace Ubc1 for poly-Ub chain extension on APC substrates. Finally, the backside Ub-binding mutant S23R-Ubc4 has no observable effect in yeast. Together, our results are consistent with a model in which Ubc4 and Ubc5 are 1) the primary E2s for Rsp5 in yeast and 2) act as monoubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human ortholog UbcH5. Ubiquitination, the process by which proteins are covalently modified by the small protein ubiquitin (Ub), 2 is a versatile method of regulation used in all eukaryotes from yeast to humans. Many cellular pathways utilize ubiquitination as a regulatory mechanism, including DNA repair, endocytosis, transcription, protein trafficking, and protein quality control (1). Ubiquitination involves a multienzyme reaction cascade in which the C terminus of Ub is first activated in an ATP-dependent reaction by the ubiquitin-activating enzyme (E1) and then transferred to the active site cysteine of a ubiquitin-conjugating enzyme (E2) to form an E2ϳUb conjugate (Fig. 1A). Ultimately, E2ϳUb transfers the Ub to a lysine side chain of a substrate in concert with a ubiquitin ligase (E3) (2). Additional Ub may be transferred to a lysine on the first Ub to form poly-Ub chains on a substrate.Protein modification by a single Ub signals different cellular fates than modification by poly-Ub chains (1). However, the determining factors for whether a substrate will be mono-or polyubiquitinated are not well understood. It is becoming increasingly clear that the E2 enzyme itself can be the determining factor for product formation catalyzed by RING (really interesting new gene)-type E3 ligases (3). For example, the human RING E3 BRCA1/BARD1 can function with 10 different E2s, and the identity of the E2 determines whether mono-or polyubiquitination is observed (4). In contrast, it is the E3 that determines the nature of the product in HECT (homologous to E6AP carboxyl-terminal ligase) E3-catalyzed Ub transfer (5).The Ubc4/UbcH5 E2 enzymes compose the largest E2 subfamily. Mos...