Ubiquitination is a post-translational modification pathway involved in myriad cellular regulation and disease pathways. The Ub (ubiquitin) 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 signalling. There are currently over 90 three-dimensional structures for 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 the present 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 and 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 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/E2∼Ub 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 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 the transfer of Ub 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 nuclear magnetic resonance and small-angle X-ray scattering. 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: the UbcH5c∼Ub conjugate 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 with regard to interaction with Ub ligases.

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 base pair scaC operator. Each ScaR subunit binds two manganese ions, and the protein is activated by a variety of other metal ions, including Cd(2+), Co(2+), and Ni(2+) but not Zn(2+). The crystal structure of apo-ScaR reveals a tertiary and quaternary structure similar to its homologue, 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 Cd(2+) and Zn(2+) 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 A away from where the ancillary site would be predicted to exist. This difference suggests that ScaR and its closely related homologues 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.