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The TFE3 and MITF master transcription factors maintain metabolic homeostasis by regulating lysosomal, melanocytic, and autophagy genes. Previous studies posited that their cytosolic retention by 14–3-3, mediated by the Rag GTPases-mTORC1, was key for suppressing transcriptional activity in the presence of nutrients. Here, we demonstrate using mammalian cells that regulated protein stability plays a fundamental role in their control. Amino acids promote the recruitment of TFE3 and MITF to the lysosomal surface via the Rag GTPases, activating an evolutionarily conserved phospho-degron leading to ubiquitination by CUL1(β-TrCP) and degradation. Elucidation of the minimal functional degron revealed a conserved alpha-helix required for interaction with RagA, illuminating the molecular basis for a severe neurodevelopmental syndrome caused by missense mutations in TFE3 within the RagA-TFE3 interface. Additionally, the phospho-degron is recurrently lost in TFE3 genomic translocations that cause kidney cancer. Therefore, two divergent pathologies converge on the loss of protein stability regulation by nutrients.

The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). We genetically introduced pairs of Cys residues in different regions of the FepA tertiary structure, with the potential to form disulfide bonds. These included Cys pairs on adjacent β-strands of the N-domain (intra-N) and Cys pairs that bridged the external surface of the N-domain to the interior of the C-terminal transmembrane β-barrel (inter-N–C). We characterized FeEnt uptake by these mutants with siderophore nutrition tests, [59Fe]Ent binding and uptake experiments, and fluorescence decoy sensor assays. The three methods consistently showed that the intra-N disulfide bonds, which restrict conformational motion within the N-domain, prevented FeEnt uptake, whereas most inter-N–C disulfide bonds did not prevent FeEnt uptake. These outcomes indicate that conformational rearrangements must occur in the N terminus of FepA during FeEnt transport. They also argue against disengagement of the N-domain out of the channel as a rigid body and suggest instead that it remains within the transmembrane pore as FeEnt enters the periplasm.

The cullin-RING ubiquitin ligases (CRLs) are ubiquitin E3 enzymes that play a key role in controlling proteasomal degradation and are activated by neddylation. We previously reported inhibitors that target CRL activation by disrupting the interaction of defective in cullin neddylation 1 (DCN1), a CRL neddylation co-E3, and UBE2M, a neddylation E2. Our first-generation inhibitors possessed poor oral bioavailability and fairly rapid clearance that hindered the study of acute inhibition of DCN-controlled CRL activity in vivo. Herein, we report studies to improve the pharmacokinetic performance of the pyrazolo-pyridone inhibitors. The current best inhibitor, 40, inhibits the interaction of DCN1 and UBE2M, blocks NEDD8 transfer in biochemical assays, thermally stabilizes cellular DCN1, and inhibits anchorage-independent growth in a DCN1 amplified squamous cell carcinoma cell line. Additionally, we demonstrate that a single oral 50 mg/kg dose sustains plasma exposures above the biochemical IC90 for 24 h in mice.

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

Chemical control of cullin neddylation is attracting increased attention based largely on the successes of the NEDD8-activating enzyme (E1) inhibitor Pevonedistat. Recently reported chemical probes enable selective and time-dependent inhibition of downstream members of the neddylation tri-enzymatic cascade including the co-E3, DCN1. In this work, we report the optimization of a novel class of small molecule inhibitors of the DCN1-UBE2M interaction. Rational X-ray co-structure enabled optimization afforded a 25-fold improvement in potency relative to the initial screening hit. The potency gains are largely attributed to additional hydrophobic interactions mimicking the N-terminal acetyl group that drives binding of UBE2M to DCN1. The compounds inhibit the protein-protein interaction, block NEDD8 transfer in biochemical assays, engage DCN1 in cells, and selectively reduce the steady-state neddylation of Cul1 and Cul3 in two squamous carcinoma cell lines harboring DCN1 amplification.

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain–containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks at their promoters. In FBXO11−/− erythroblasts, these gene promoters bind BAHD1 and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2, and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at many developmentally poised bivalent genes during erythropoiesis.

RING E3s comprise the largest family of ubiquitin (UB) and ubiquitin-like protein (UBL) ligases. RING E3s typically promote UB or UBL transfer from the active site of an associated E2 enzyme to a distally-recruited substrate. Many RING E3s - including the cullin-RING ligase family - are multifunctional, interacting with various E2s (or other E3s) to target distinct proteins, transfer different UBLs, or to initially modify substrates with UB or subsequently elongate UB chains. Here we consider recent structures of cullin-RING ligases, and their partner E2 enzymes, representing ligation reactions. The studies collectively reveal multimodal mechanisms - interactions between ancillary E2 or E3 domains, post-translational modifications, or auxiliary binding partners - directing cullin-RING E3-E2 enzyme active sites to modify their specific targets.

The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). We genetically introduced pairs of Cys residues in different regions of the FepA tertiary structure, with the potential to form disulfide bonds. These included Cys pairs on adjacent β-strands of the N-domain (intra-N) and Cys pairs that bridged the external surface of the N-domain to the interior of the C-terminal transmembrane β-barrel (inter-N–C). We characterized FeEnt uptake by these mutants with siderophore nutrition tests, [(59)Fe]Ent binding and uptake experiments, and fluorescence decoy sensor assays. The three methods consistently showed that the intra-N disulfide bonds, which restrict conformational motion within the N-domain, prevented FeEnt uptake, whereas most inter-N–C disulfide bonds did not prevent FeEnt uptake. These outcomes indicate that conformational rearrangements must occur in the N terminus of FepA during FeEnt transport. They also argue against disengagement of the N-domain out of the channel as a rigid body and suggest instead that it remains within the transmembrane pore as FeEnt enters the periplasm.

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure-activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1-UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1-UBE2M protein-protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers.

Many fundamental discoveries in ubiquitin-proteasome research have relied on reconstitution of activities from purified or recombinantly expressed components. These include landmark discoveries of E1-E2-E3 mechanisms, in which ubiquitin (UB) is initially activated and then covalently shuttled between enzyme active sites and ultimately ligated to substrate or substrate-linked UBs during polyubiquitination. However, recent studies have unearthed enormous variations on the E1-E2-E3 theme; for example, one E3 may employ two distinct E2s, or two different E3s may act in a single assembly or in series, to prime substrates directly with UB and subsequently decorate them with myriad types of polyubiquitin chains. To dissect this complexity, it can be helpful to monitor specific UB transfer reactions in isolation, rather than the end-point products formed upon mixing all enzymes in a cascade. Pulse-chase assays enable observation of a single reaction step and also allow one to differentially label UBs carried by different enzymes within the same tube. In such assays, the "pulse" reaction generates a thioester-linked enzyme~UB intermediate, while the "chase" monitors UB transfer to downstream components over time. Here, we describe pulse-chase assays for detecting fluorescent-UB in E2~UB intermediates. These assays enable direct assessment of particular ligation reactions, alone and in combination, to explore roles of multiple enzymatic cascades in the same tube. We anticipate this technique can be adapted to many different E2s, as well as thioester-forming E3s, to dissect ubiquitination by many distinct enzyme cascades.

Many fundamental discoveries in ubiquitin-proteasome research have relied on reconstitution of activities from purified or recombinantly expressed components. These include landmark discoveries of E1-E2-E3 mechanisms, in which ubiquitin (UB) is initially activated and then covalently shuttled between enzyme active sites and ultimately ligated to substrate or substrate-linked UBs during polyubiquitination. However, recent studies have unearthed enormous variations on the E1-E2-E3 theme; for example, one E3 may employ two distinct E2s, or two different E3s may act in a single assembly or in series, to prime substrates directly with UB and subsequently decorate them with myriad types of polyubiquitin chains. To dissect this complexity, it can be helpful to monitor specific UB transfer reactions in isolation, rather than the end-point products formed upon mixing all enzymes in a cascade. Pulse-chase assays enable observation of a single reaction step and also allow one to differentially label UBs carried by different enzymes within the same tube. In such assays, the “pulse” reaction generates a thioester-linked enzyme ~ UB intermediate, while the “chase” monitors UB transfer to downstream components over time. Here, we describe pulse-chase assays for detecting fluorescent-UB in E2 ~ UB intermediates. These assays enable direct assessment of particular ligation reactions, alone and in combination, to explore roles of multiple enzymatic cascades in the same tube. We anticipate this technique can be adapted to many different E2s, as well as thioester-forming E3s, to dissect ubiquitination by many distinct enzyme cascades.

We previously reported the discovery, validation, and structure–activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1–UBE2M interaction. We demonstrated that compound 7 inhibits both the DCN1–UBE2M protein–protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound 7 exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CLint = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (67). Compound 67 retains the favorable biochemical and cellular activity of compound 7 but is significantly more stable both in vitro and in vivo. Compound 67 is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1–UBE2M interaction on the NEDD8/CUL pathway.

SCF ubiquitin ligase assembly is regulated by the interplay of substrate binding, reversible Nedd8 conjugation on Cul1, and the F-box protein (FBP) exchange factors Cand1 and Cand2. Detailed investigations into SCF assembly and function in reconstituted systems and Cand1,2 knockout cells informed the development of a mathematical model for how dynamical assembly of SCF complexes is controlled, and how this cycle is coupled to degradation of an SCF substrate. Simulations predicted an unanticipated hypersensitivity of Cand1/2-deficient cells to FBP expression levels, which was experimentally validated. Together, these and prior observations lead us to propose the adaptive exchange hypothesis, which posits that regulation of the koff of an FBP from SCF by the actions of substrate, Nedd8, and Cand1 molds the cellular repertoire of SCF complexes, and that the plasticity afforded by this exchange mechanism may enable large variations in FBP expression during development and in FBP gene number during evolution.

The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKβ. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated.

Mature red blood cells (RBC) contain approximately 95% cytosolic hemoglobin for the purpose of blood oxygen transport. This specialized state is achieved during erythropoiesis by regulated gene expression and protein degradation. During late-stage erythropoiesis, ubiquitin ligases eliminate unnecessary proteins and maintain quality control by degrading unstable proteins, including unpaired hemoglobin subunits. However, ubiquitin ligases are expressed at all stages of erythropoiesis and the functions of most are unknown. To study ubiquitin ligases involved in RBC formation, we performed a Cas9/single guide (sg) RNA screen for functional ubiquitin-proteasome components in HUDEP-2 cells, an immortalized human cell line that proliferates as immature erythroblasts and can be induced to undergo terminal maturation. We identified the E3 ubiquitin ligase FBXO11 as a top-ranked candidate. FBXO11 is a member of the F-box protein family that assembles into a SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. Depletion of FBXO11 by 2 different sgRNAs in HUDEP-2 cells inhibited erythroid maturation, as evidenced by reduced hemoglobinization, failure to induce the maturation marker Band3 and persistence of immature cell morphology. In primary human CD34+ cells, suppression of FBXO11 expression by Cas9 + two independent sgRNAs inhibited erythroid maturation, as evidenced by reduced Band3 expression (5.3% vs. 15.6% for non-targeting sgRNA, P < 0.001; n=4). RNA-seq analysis of FBXO11-depleted HUDEP-2 cells revealed 951 decreased transcripts (enriched for erythroid genes) and 339 increased transcripts (enriched for genes expressed in activated T-cells) compared to control cells expressing non-targeting gRNA (P < 0.05). Thus, FBXO11 is required for erythroid maturation and facilitates erythroid gene expression. We sought to establish how FBXO11 modulates erythropoiesis and erythroid gene expression by identifying the relevant ubiquitination substrate(s). Combined quantitative proteome analysis with RNA-seq of FBXO11-depleted HUDEP-2 cells identified several proteins that are upregulated with no change in their corresponding mRNA. We tested whether reduction of these candidate substrates could alleviate the erythroid maturation block conferred by FBXO11 depletion. In FBXO11 gene-disrupted HUDEP-2 cells, suppression of the heterochromatin-associated protein BAHD1 partially rescued hemoglobinization and Band3 expression (4.2% for Cas9 + non-targeting sgRNA vs. 21.7% for Cas9 + BAHD1 sgRNAs, P < 0.01; n=3) . Conversely, stable overexpression of V5-epitope-tagged BAHD1 in WT HUDEP-2 cells reduced Band3 expression from 25.0% to 11.4% (P < 0.001; n=3) and inhibited hemoglobinization. Transcriptome analysis demonstrated a significant inverse correlation between genes deregulated by BAHD1-V5 overexpression and FBXO11 deficiency in HUDEP-2 cells, particularly for erythroid genes that are downregulated (P < 0.0001). BAHD1, named after its bromo-adjacent homology domain that interacts with H3K27me3, is part of a transcriptional repressor complex. We showed that BAHD1 and FBXO11 co-immunoprecipitated in cells and that BAHD1 amino (N)-terminal segments of 188 or 240 amino acids were robustly modified with ubiquitin by SCFFBXO11 complex. Chromatin immunoprecipitation-sequencing analysis of BAHD1-V5-expressing WT HUDEP-2 cells showed strong enrichment for BAHD1 occupancy on erythroid gene promoters that were downregulated by FBXO11-deficiency (P < 0.0001). We next investigated whether a specific set of histone marks distinguish FBXO11-regulated genes in normal erythroblasts. We found that most FBXO11-regulated genes in both HUDEP-2 and primary CD34+ derived erythroblasts harbor histone marks H3K4me3 and H3K27me3, indicating a "bivalent" epigenetic state that supports low level transcription in stem or progenitor cells. Together, these data indicate that FBXO11 activates expression of erythroid genes by ubiquitinating and degrading bivalent promoter-bound BAHD1 repressor complexes with likely resolution to a monovalent transcriptionally active state. Overall, our findings identify FBXO11 as a ubiquitin ligase that utilizes a novel mechanism to activate erythroid genes during RBC formation. This newly identified pathway may contribute to known activities of FBXO11 as a tumor suppressor and developmental regulator in non-erythroid tissues. Disclosures No relevant conflicts of interest to declare.

In ubiquitin-like protein (UBL) cascades, a thioester-linked E2∼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53's WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12∼Rub1. Dcn1's "potentiating neddylation" domain (Dcn1(P)) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12∼Rub1 discharge and directing Ubc12's active site to Cdc53. Crystal structures of Dcn1(P)-Cdc53(WHB) and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1's interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12∼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.

In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2 approximately Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5B approximately Ub). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.

Cullin-based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit by the ubiquitin-like protein Nedd8. Cullin neddylation dissociates the ligase assembly inhibitor Cand1, and promotes E2 recruitment and ubiquitin transfer by inducing a conformational change. Here, we have identified and characterized Lag2 as a likely Saccharomyces cerevisiae orthologue of mammalian Cand1. Similar to Cand1, Lag2 directly interacts with non-neddylated yeast cullin Cdc53 and prevents its neddylation in vivo and in vitro. Binding occurs through a conserved C-terminal beta-hairpin structure that inserts into the Skp1-binding pocket on the cullin, and an N-terminal motif that covers the neddylation lysine. Interestingly, Lag2 is itself neddylated in vivo on a lysine adjacent to this N-terminal-binding site. Overexpression of Lag2 inhibits Cdc53 activity in strains defective for Skp1 or neddylation functions, implying that these activities are important to counteract Lag2 in vivo. Our results favour a model in which binding of substrate-specific adaptors triggers release of Cand1/Lag2, whereas subsequent neddylation of the cullin facilitates the removal and prevents re-association of Lag2/Cand1.

Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation.

Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin–proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.

We created hybrid proteins to study the functions of TonB. We first fused the portion ofEscherichia coli tonBthat encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream fromE. coli malE(MalE-TonB69C). Production of MalE-TonB69C intonB+bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus toE. coliYcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

How RING E3 ligases mediate E2-to-substrate ubiquitin-like protein (UBL) transfer remains unknown. Here we address how the RING E3 RBX1 positions NEDD8's E2 (UBC12) and substrate (CUL1). We find that existing structures are incompatible with CUL1 NEDD8ylation and report a new conformation of RBX1 that places UBC12 adjacent to CUL1. We propose RING domain rotation as a general mechanism for UBL transfer for the largest family of E3s.

SummaryMost E3 ligases use a RING domain to activate a thioester-linked E2∼ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2∼UBL-target intermediate representing RBX1-UBC12∼NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2∼UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RING-E2∼UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.

The periplasmic protein FepB ofEscherichia coliis a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay theKdof the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (Kd= 30 nM) and ferric enantioenterobactin (Kd= 15 nM), the left-handed stereoisomer of the naturalE. colisiderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (Kd= 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

The siderophore ferric enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein. After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (Kd) and uptake (Km): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric enterobactin: the interaction between the protein and the ferric siderophore is so avid (Kd < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric enterobactin transport occurred without them, at levels that allowed bacterial growth.

Little is known about molecular recognition of acetylated N-termini, despite prevalence of this modification among eukaryotic cytosolic proteins. We report that the family of human DCN-like (DCNL) co-E3s, which promote ligation of the ubiquitin-like protein NEDD8 to cullin targets, recognizes acetylated N-termini of the E2 enzymes UBC12 and UBE2F. Systematic biochemical and biophysical analyses reveal 40- and 10- fold variations in affinities amongst different DCNL-cullin and DCNL-E2 complexes, which contribute to widely ranging efficiencies of different NEDD8 ligation cascades. Structures of DCNL2 and DCNL3 complexes with N-terminally acetylated peptides from UBC12 and UBE2F illuminate a common mechanism by which DCNL proteins recognize N-terminally acetylated E2s, and how selectivity for interactions dependent on N-acetyl-methionine can be established through sidechains recognizing distal residues. Distinct preferences of UBC12 and UBE2F peptides for inhibiting different DCNLs, including the oncogenic DCNL1 protein, suggest it may be possible to develop small molecules blocking specific N-acetyl-methionine-dependent protein interactions.

Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation.

Ubiquitination of protein is well known mechanism for the regulation of cellular proteins by the ubiquitin-proteasome system (UPS). The ubiquitin-proteasome system (UPS) plays a crucial role in regulating cellular protein degradation and turnover. Dysregulation of the UPS has been associated with a wide variety of diseases including cancers, neurodegenerative disorders, and viral and bacterial infections. Recently, there are emerging evidences show that targeting this UPS by small molecule modulator is a useful anticancer strategy based on the previous successes of proteasome inhibitors, E3 inhibitors and the NEDD8 E1 inhibitor. In particular, we are targeting NEDD8 ligation to cullins via inhibition of DCN1 as a small molecule target. DCN1 was known to interacts with cullins and is required for neddylation in vivo. Furthermore, dysregulation of DCN1 is associated with a number of human cancers. DCN1 activity is controlled by its interaction with UBC12. Therefore, we have developed biochemical assay utilizing TR-FRET technology for the purpose of high-throughput screening (HTS) to identify the small molecule inhibitors that disrupt the DCN1-UBC12 protein-protein interaction and use them to elucidate the biological effects of DCN1 mediated UPS regulation. Herein, we report our progress including design, procedure and result of HTS with post-screen analysis to discover small molecule inhibitors of the DCN1 from our in-house library compounds (∼600K). Following hit identification and validation, we identified three novel scaffolds as novel DCN1 inhibitors by biochemical, biophysical, and crystallographic studies to follow for further hit optimization. To the best of our knowledge, this is the first work to report small molecule DCN1 inhibitors. Citation Format: Jaeki Min, Daniel C. Scott, Sergio C. Chai, Asli N. Goktug, Jared T. Hammill, Guochang Huang, Michele C. Connelly, Amy Matheny, Anang Shelat, Bhuvanesh Singh, Taosheng Chen, Brenda A. Schulman, R. Kiplin Guy. Discovery of novel small molecule inhibitors of the DCN1-Ubc12 interaction by high throughput screening technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-056.

Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.

Cullin-RING ligases (CRLs) compose the largest class of E3 ubiquitin ligases. CRLs are modular, multisubunit enzymes, comprising interchangeable substrate receptors dedicated to particular Cullin-RING catalytic cores. Recent structural studies have revealed numerous ways in which CRL E3 ligase activities are controlled, including multimodal E3 ligase activation by covalent attachment of the ubiquitin-like protein NEDD8, inhibition of CRL assembly/activity by CAND1, and several mechanisms of regulated substrate recruitment. These features highlight the potential for CRL activities to be tuned in responses to diverse cellular cues, and for modulating CRL functions through small-molecule agonists or antagonists. As the second installment of a two-review series, this article focuses on recent structural studies advancing our knowledge of how CRL activities are regulated.

Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops ofEscherichia coliFepA during its transport of the siderophore ferric enterobactin. Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein. The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2. Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332. These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 Å, from a ligand-free open state to a ligand-bound closed state. The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanidem-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty beta-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.

SummaryCullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8∼Cul5ctd-Rbx1 and SAXS analysis of NEDD8∼Cul1ctd-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.

Skp1-Cul1-Fbox (SCF) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCFSkp2/Cks1 and SCFFbw7 and found that CSN occludes both SCF functional sites—the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination., Cell Reports, 2 (3), ISSN:2666-3864, ISSN:2211-1247

The content of molybdenum in wild-type and chlD cells was measured under a variety of growth conditions to determine if cells with a defective chlD gene were able to accumulate molybdenum. The chlD cells accumulated less molybdenum than wild-type cells did but concentrated molybdenum to a level at least 20-fold higher than the concentration in the culture medium. Molybdenum was present within spheroplasts of chlD cells and was not dialyzable. The chlD cells accumulated as much molybdenum as wild-type cells did when grown in medium containing 0.1 mM molybdate; thus, the capability of incorporation of molybdenum into cellular component(s) was equivalent to that of the wild type under these conditions.

Labeled nucleic acid probes are used as diagnostic tools by detecting changes in gene expression upon hybridization to target RNAs or DNAs that are related to specific disease genes. 5-[S-(2,4-Dinitrophenyl)-thio]-2′-deoxyuridine analog represents an excellent nucleic acid label, containing the DNP group which functions both as a probe and as a precursor for the introduction of a variety of fluorescent groups. This study describes thermal denaturation hybridization experiments with oligonucleotides containing the 5-[S-(2,4-dinitrophenyl)-thio]-2′-deoxyuridine analog. Using molecular modeling techniques, the effects of this analog on the hybrid structure and stability were examined, including (i) analog conformation, (ii) hydrogen bonding, (iii) stacking interactions and (iv) hybrid helical geometry. This analog does not prohibitively affect the hybrid thermal stability and incorporation of the analog does not compromise the structural integrity of the double helix. In particular, the sequence-dependence of the analog effects and the dependence on the modification site relative to the end(s) of the helix were investigated. Findings described here should provide guidelines in the rational design of nucleic acid probes. Copyright © 1999 John Wiley & Sons, Ltd.

Summary Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.