Abstract Cellular responses to amino acid fluctuations often hinge on ubiquitin-mediated control of metabolic enzymes, yet the underlying E3 ligase pathways remain poorly defined. Using quantitative proteomics and active cullin-RING ligase (CRL) profiling, we identify LRRC58 as a cysteine-responsive substrate receptor whose stability increases sharply under cysteine starvation. Proteomics reveals an inverse relationship between LRRC58 and the metabolic enzyme cysteine dioxygenase 1 (CDO1), suggesting a cysteine-linked regulatory axis. Biochemical reconstitution and cryo-EM structures show that LRRC58 forms an active CUL2- or CUL5-based CRL that selectively positions CDO1 for ubiquitylation at Lys8. Disease mutant versions of CDO1 mapping to the LRRC58 interface and impaired for the endogenous ubiquitylation pathway were degraded through orthogonal targeting by a VHL-based degrader. Together, our proteomics-guided discovery pipeline, cellular stability studies, and structural analyses uncover a metabolically-tuned LRRC58-CDO1 pathway that links cysteine availability to selective proteasomal turnover, reveals principles of metabolite-regulated CRL activity, and showcases mechanisms distinguishing endogenous and targeted protein degradation.

Ubiquitin (Ub) is a protein post-translational modifier that controls proteostasis through mechanisms spanning transcription, translation, and protein degradation. Ub conjugation occurs through a cascade of three enzyme classes (E1, E2, and E3s) involving >1,000 proteins that regulate the ubiquitination of cellular proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized. Here, we develop highly selective and potent pan-UBE2D/UBCH5 inhibitors by targeting the RING- and backside-binding sites with engineered linked-domain proteins. In HeLa cells, these inhibitors phenocopy the knockdown of UBE2D by enhancing chemosensitivity to cisplatin. Whole-cell proteomics reveals that ∼20% of the identified proteins are more abundant, and most do not have altered mRNA levels, suggesting that their protein turnover is regulated by UBE2D. Enrichment analysis of the altered mRNAs indicates that the linked-domain proteins trigger the unfolded protein response. These precision tools will enable new studies probing UBE2D’s cellular roles and help to deconvolute complex Ub regulatory networks.

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2’s intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.

E3 ubiquitin ligases, in collaboration with E2 ubiquitin-conjugating enzymes, modify proteins with poly-ubiquitin chains. Cullin-RING ligase (CRL) E3s use Cdc34/UBE2R-family E2s to build Lys48-linked poly-ubiquitin chains to control an enormous swath of eukaryotic biology. Yet the molecular mechanisms underlying this exceptional linkage specificity and millisecond kinetics of poly-ubiquitylation remain unclear. Here we obtain cryogenic-electron microscopy (cryo-EM) structures that provide pertinent insight into how such poly-ubiquitin chains are forged. The CRL RING domain not only activates the E2-bound ubiquitin but also shapes the conformation of a distinctive UBE2R2 loop, positioning both the ubiquitin to be transferred and the substrate-linked acceptor ubiquitin within the active site. The structures also reveal how the ubiquitin-like protein NEDD8 uniquely activates CRLs during chain formation. NEDD8 releases the RING domain from the CRL, but unlike previous CRL–E2 structures, does not contact UBE2R2. These findings suggest how poly-ubiquitylation may be accomplished by many E2s and E3s.

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of ~20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.

AbstractSpecificity of the ubiquitin-proteasome system depends on E3 ligase-substrate interactions. Many such pairings depend on E3 ligases binding to peptide-like sequences - termed N- or C-degrons - at the termini of substrates. However, our knowledge of structural features distinguishing closely related C-degron substrate-E3 pairings is limited. Here, by systematically comparing ubiquitylation activities towards a suite of common model substrates, and defining interactions by biochemistry, crystallography, and cryo-EM, we reveal principles of C-degron recognition across the KLHDCX family of Cullin-RING ligases (CRLs). First, a motif common across these E3 ligases anchors a substrate’s C-terminus. However, distinct locations of this C-terminus anchor motif in different blades of the KLHDC2, KLHDC3, and KLHDC10 β-propellers establishes distinct relative positioning and molecular environments for substrate C-termini. Second, our structural data show KLHDC3 has a pre-formed pocket establishing preference for an Arg or Gln preceding a C-terminal Gly, whereas conformational malleability contributes to KLHDC10’s recognition of varying features adjacent to substrate C-termini. Finally, additional non-consensus interactions, mediated by C-degron binding grooves and/or by distal propeller surfaces and substrate globular domains, can substantially impact substrate binding and ubiquitylatability. Overall, the data reveal combinatorial mechanisms determining specificity and plasticity of substrate recognition by KLDCX-family C-degron E3 ligases.

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.

Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation. Given the homology between HOIL-1 and ARIH1, an RBR E3 that functions with the large superfamily of cullin-RING E3 ligases (CRLs), a biochemical investigation was undertaken, showing ARIH1 catalyzes Ser ubiquitylation to CRL-bound substrates. However, the efficiency of ubiquitylation was exquisitely dependent on the location and chemical environment of the Ser residue within the primary structure of the substrate. Comprehensive mutagenesis of the ARIH1 Rcat domain identified residues whose mutation severely impacted both oxyester and isopeptide bond formation at the preferred site for Ser ubiquitylation while only modestly affecting Lys ubiquitylation at the physiological site. The results reveal dual isopeptide and oxyester protein ubiquitylation activities of ARIH1 and set the stage for physiological investigations into this function of emerging importance.

Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes.

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates 1 , 2 . However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies 3 – 7 . Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation.

Eukaryotic cell biology depends on cullin–RING E3 ligase (CRL)-catalysed protein ubiquitylation1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD82–6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1β-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin–RING proteins. A cryo-electron microscopy structure provides insights into the activation of cullin–RING E3 ligases by NEDD8 and the consequent catalysis of ubiquitylation reactions.

Perhaps in a decade or so, researchers may look back on current times and view it as an inflection point for the development of novel therapeutic modalities for the treatment of various human diseases. One particularly innovative approach involves targeting disease-modifying proteins for degradation by the proteasome, the large multisubunit protease responsible for the lion's share of regulated protein degradation in human cells (1). The technique uses the action of ubiquitin (UB) ligases, the enzymes that append polyubiquitin chains onto proteins that signal for degradation, and unleashes their activities on disease-causing proteins. Inducing proximity between the ubiquitin ligase and the target is accomplished by small molecule effectors that bind in shallow grooves on both the enzyme and the protein target, acting as “glue” and driving substantial affinities between two proteins that normally do not interact in cells (2, 3). While this may sound more like a futuristic treatment than reality, there are several Food and Drug Administration-approved therapies utilizing induced proximity to target proteins to the proteasome, and even more are in clinical trials (4). While one concern had been that molecular glues capable of promoting protein–protein interactions are quite rare, the study in PNAS by Lin et al. (5) is likely to change opinions on the matter. Before explaining the results from Lin et al. (5), a bit of background is necessary. Most of the current therapeutic molecular glues that target disease-causing proteins for degradation utilize the largest family of ubiquitin ligases in humans, the Cullin-RING (Really Interesting New Gene) ligases (CRLs) (6, 7). Some 200 of these enzymes can be found in our cells and account for at least 20% of all proteasome-dependent protein degradation. This grand number of enzymes associates with seven Cullin scaffold proteins, which bind to RING proteins (RBX1/2) that recruit ubiquitin-carrying enzymes. … [↵][1]1To whom correspondence may be addressed. Email: danny.scott{at}stjude.org. [1]: #xref-corresp-1-1

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors—the lysine side chain—affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.

Protein misfolding is a recurring phenomenon that cells must manage; otherwise misfolded proteins can aggregate and become toxic should they persist. To counter this burden, cells have evolved protein quality control (PQC) mechanisms that manage misfolded proteins. Two classes of systems that function in PQC are chaperones that aid in protein folding and ubiquitin-protein ligases that ubiquitinate misfolded proteins for proteasomal degradation. How folding and degradative PQC systems interact and coordinate their respective functions is not yet fully understood. Previous studies of PQC degradation pathways in the endoplasmic reticulum and cytosol have led to the prevailing idea that these pathways require the activity of Hsp70 chaperones. Here, we find that involvement of the budding yeast Hsp70 chaperones Ssa1 and Ssa2 in nuclear PQC degradation varies with the substrate. In particular, nuclear PQC degradation mediated by the yeast ubiquitin-protein ligase San1 often involves Ssa1/Ssa2, but San1 substrate recognition and ubiquitination can proceed without these Hsp70 chaperone functions in vivo and in vitro. Our studies provide new insights into the variability of Hsp70 chaperone involvement with a nuclear PQC degradation pathway.

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

Ubiquitin ligases (E3s) function by binding to both a protein substrate and to ubiquitin-conjugating enzymes (E2s) bound to ubiquitin. E3s facilitate the transfer of ubiquitin from the E2 active site to an E3-bound substrate. Thus, the affinity of the interaction of an E2 with its E3 partner is of considerable interest. The purpose of this work is to (1) provide protocols for the purification of the human E2 Cdc34, as well as for some additional protein components needed for the assays described here whose purification protocols haven't been described elsewhere in detail; (2) provide the researcher with critical information regarding the proper long-term storage of these enzymes to retain maximal activity; (3) provide a protocol to benchmark Cdc34 activity with previously described activity levels in the literature; and (4) provide a simple and rapid means of measuring E2 affinity for an E3.