A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters

Cells rapidly remodel their proteomes to align their cellular metabolism to environmental conditions. Ubiquitin E3 ligases enable this response, by facilitating rapid and reversible changes to protein stability, localization, or interaction partners. In Saccharomyces cerevisiae, the GID E3 ligase regulates the switch from gluconeogenic to glycolytic conditions through induction and incorporation of the substrate receptor subunit Gid4, which promotes the degradation of gluconeogenic enzymes. Here, we show an alternative substrate receptor, Gid10, which is induced in response to changes in temperature, osmolarity, and nutrient availability, regulates the ART‐Rsp5 ubiquitin ligase pathway, a component of plasma membrane quality control. Proteomic studies reveal that the levels of the adaptor protein Art2 are elevated upon GID10 deletion. A crystal structure shows the basis for Gid10‐Art2 interactions, and we demonstrate that Gid10 directs a GID E3 ligase complex to ubiquitinate Art2. Our data suggest that the GID E3 ligase affects Art2‐dependent amino acid transport. This study reveals GID as a system of E3 ligases with metabolic regulatory functions outside of glycolysis and gluconeogenesis, controlled by distinct stress‐specific substrate receptors.     

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4‐1) and Nedd4L (Nedd4‐2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4‐1 and Nedd4‐2, and rat‐Nedd4‐1, using protein microarrays spotted with ～8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4‐1 and Nedd4‐2, others were specific to only one, with several Tyr kinases preferred by Nedd4‐1 and some ion channels by Nedd4‐2; this was subsequently validated in vivo. Accordingly, Nedd4‐1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4‐1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.     

The mammalian N-end rule pathway: new insights into its components and physiological roles

The N-end rule pathway is a ubiquitin-dependent proteolytic system, in which destabilizing N-terminal residues of short-lived proteins function as an essential determinant of an N-terminal degradation signal (N-degron). An N-degron can be created from a pre-N-degron through specific N-terminal modifications, providing a means conditionally to destabilize otherwise stable polypeptides. The pathway has been found in all organisms examined, from prokaryotes to eukaryotes. Recent biochemical and proteomic studies identified many components of the mammalian N-end rule pathway, including a family of substrate recognition ubiquitin ligases and their substrates. The genetic dissection in animals and humans revealed its essential role in various vital physiological processes, ranging from cardiovascular development and meiosis to the pathogenesis of human genetic diseases. These discoveries have provided new insights into the components, functions and mechanics of this unique proteolytic system.     

The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies

The N-end rule states that the half-life of a protein is determined by the nature of its N-terminal residue. This fundamental principle of regulated proteolysis is conserved from bacteria to mammals. Although prokaryotes and eukaryotes employ distinct proteolytic machineries for degradation of N-end rule substrates, recent findings indicate that they share common principles of substrate recognition. In eukaryotes substrate recognition is mediated by N-recognins, a class of E3 ligases that labels N-end rule substrates via covalent linkage to ubiquitin, allowing the subsequent substrate delivery to the 26S proteasome. In bacteria, the adaptor protein ClpS exhibits homology to the substrate binding site of N-recognin. ClpS binds to the destabilizing N-termini of N-end rule substrates and directly transfers them to the ClpAP protease.     

The E3 Ubiquitin Ligase Atrophin Interacting Protein 4 Binds Directly To The Chemokine Receptor CXCR4 Via a Novel WW Domain-mediated Interaction

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.     

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.     

How are substrates recognized by the ubiquitin-mediated proteolytic system

A necessary step in the degradation of proteins by the ubiquitin system is recognition by the ubiquitin-protein ligases(s). Various structural features of the proteolytic substrate render it susceptible to conjugation with ubiquitin. The N-terminal residue plays a major role in this process, with distinct sites on the ligase(s) recognizing specific types of N-termini. Post-translational modification of some of these residues is required prior to their recognition. A free N terminus is not the only marker; proteins with either free or blocked N termini can be recognized via structural domains that are downstream and distinct from this residue.     

Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases

The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.     

Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.     

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation

Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.     

Recognition of the gluconeogenic enzyme,Pck1, via the Gid4 E3 ligase: An in silico perspective

Gluconeogenesis, the reverse process of glycolysis, is a favorable mechanism at conditions of glucose deprivation. Pck1 is a rate‐limiting gluconeogenic enzyme, where its deficiency or mutation contributes to serious clinical situations as neonatal hypoglycemia and liver failure. A recent report confirms that Pck1 is a target for proteasomal degradation through its proline residue at the penultimate position, recognized by Gid4 E3 ligase, but with a lack of informative structural details. In this study, we delineate the localized sequence motif, degron, that specifically interact with Gid4 ligase and unravel the binding mode of Pck1 to the Gid4 ligase by using molecular docking and molecular dynamics. The peptide/protein docking HPEPDOCK web server along with molecular dynamic simulations are applied to demonstrate the binding mode and interactions of a Pck1 wild type (SPSK) and mutant (K4V) with the recently solved structure of Gid4 ligase. Results unveil a distinct binding mode of the mutated peptide compared with the wild type despite having comparable binding affinities to Gid4. Moreover, the four‐residue peptide is found insufficient for Gid4 binding, while the seven‐residue peptide suffices for binding to Gid4. The amino acids S134, K135, and N137 in the loop L1 (between β1 and β2) of the Gid4 are essential for the stabilization of the seven‐residue peptide in the binding site of the ligase. The presence of Val⁴ instead of Lys⁴ smashes the H‐bonds that are formed between Lys⁴ and Gid4 in the wild type peptide, making the peptide prone to bind with the other side of the binding pocket (L4 loop of Gid4). The dynamics of Gid4 L3 loop is affected dramatically once K4V mutant Pck1 peptide is introduced. This opens the door to explore the mutation effects on the binding mode and smooth the path to target protein degradation by design competitive and non‐competitive inhibitors.     

Distinct functional domains of Ubc9 dictate cell survival and resistance to genotoxic stress

Covalent modification with SUMO alters protein function, intracellular localization, or protein-protein interactions. Target recognition is determined, in part, by the SUMO E2 enzyme, Ubc9, while Siz/Pias E3 ligases may facilitate select interactions by acting as substrate adaptors. A yeast conditional Ubc9P(123)L mutant was viable at 36 degrees C yet exhibited enhanced sensitivity to DNA damage. To define functional domains in Ubc9 that dictate cellular responses to genotoxic stress versus those necessary for cell viability, a 1.75-A structure of yeast Ubc9 that demonstrated considerable conservation of backbone architecture with human Ubc9 was solved. Nevertheless, differences in side chain geometry/charge guided the design of human/yeast chimeras, where swapping domains implicated in (i) binding residues within substrates that flank canonical SUMOylation sites, (ii) interactions with the RanBP2 E3 ligase, and (iii) binding of the heterodimeric E1 and SUMO had distinct effects on cell growth and resistance to DNA-damaging agents. Our findings establish a functional interaction between N-terminal and substrate-binding domains of Ubc9 and distinguish the activities of E3 ligases Siz1 and Siz2 in regulating cellular responses to genotoxic stress.     

Eotaxin and monocyte chemotactic protein-3 use different modes of action

Eotaxin selectively binds CC chemokine receptor (CCR) 3, whereas monocyte chemotactic protein (MCP)-3 binds CCR1, CCR2, and CCR3. To identify the functional determinants of the chemokines, we generated four reciprocal chimeric chemokines-M10E9, M22E21, E8M11, and E20M23-by shuffling the N-terminus and N-loop of eotaxin and MCP-3. M22E21 and E8M11, which shared the N-loop from MCP-3, bound to monocytes with high affinity, and activated monocytes. In contrast, M10E9 and E20M23, which lacked the N-loop, failed to bind and transduce monocyte responses, identifying the N-loop of MCP-3 as the selectivity determinant for CCR1/CCR2. A BIAcore assay with an N-terminal peptide of CCR3 (residues 1-35) revealed that all chimeras except E20M23 exhibited varying degrees of binding affinity with commensurate chemotaxis activity of eosinophils. Surprisingly, E20M23 could neither bind the CCR3 peptide nor activate eosinophils, despite having both N-terminal motifs from eotaxin. These results suggest that the two N-terminal motifs of eotaxin must cooperate with other regions to successfully bind and activate CCR3.     

Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A

Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.     

Multisite phosphorylation and binding alter conformational dynamics of the 4E-BP2 protein

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamic characterization of their ensembles remain challenging, both in isolation and when they form dynamic “fuzzy” complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Förster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a non-uniform segmental flexibility around six different labeling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-to-microsecond timescales. Upon hyperphosphorylation, which induces folding of ～40 residues in 4E-BP2, the quenching rates decreased at most labeling sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs significantly increased upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step toward a mechanistic understanding of this important IDP via integrative modeling.     

Exploring the topology of the Gid complex, the E3 ubiquitin ligase involved in catabolite-induced degradation of gluconeogenic enzymes

In the yeast Saccharomyces cerevisiae, key regulatory enzymes of gluconeogenesis such as fructose-1,6-bisphosphatase are degraded via the ubiquitin proteasome system when cells are replenished with glucose. Polyubiquitination is carried out by the Gid complex, a multisubunit ubiquitin ligase that consists of seven different Gid (glucose-induced degradation-deficient) proteins. Under gluconeogenic conditions the E3 ligase is composed of six subunits (Gid1/Vid30, Gid2/Rmd5, Gid5/Vid28, Gid7, Gid8, and Gid9/Fyv10). Upon the addition of glucose the regulatory subunit Gid4/Vid24 appears, binds to the Gid complex, and triggers ubiquitination of fructose-1,6-bisphosphatase. All seven proteins are essential for this process; however, nothing is known about the arrangement of the subunits in the complex. Interestingly, each Gid protein possesses several remarkable motifs (e.g. SPRY, LisH, CTLH domains) that may play a role in protein-protein interaction. We, therefore, generated altered versions of individual Gid proteins by deleting or mutating these domains and performed co-immunoprecipitation experiments to analyze the interaction between distinct subunits. Thus, we were able to create an initial model of the topology of this unusual E3 ubiquitin ligase.     

GroEL recognises sequential and non-sequential linear structural motifs compatible with extended beta-strands and alpha-helices.

The chaperonin GroEL binds a variety of polypeptides that share no obvious sequence similarity. The precise structural, chemical and dynamic features that are recognised remain largely unknown. Structural models of the complex between GroEL and its co-chaperonin GroES, and of the isolated apical domain of GroEL (minichaperone; residues 191-376) with a 17 residue N-terminal tag show that a linear sequential sequence (extended beta-strand) can be bound. We have analysed characteristics of the motifs that bind to GroEL by using affinity panning of immobilised GroEL minichaperones for a library of bacteriophages that display the fungal cellulose-binding domain of the enzyme cellobiohydrolase I. This protein has seven non-sequential residues in its binding site that form a linear binding motif with similar dimensions and characteristics to the peptide tag that was bound to the minichaperone GroEL(191-376). The seven residues thus form a constrained scaffold. We find that GroEL does bind suitable mutants of these seven residues. The side-chains recognised do not have to be totally hydrophobic, but polar and positively charged chains can be accommodated. Further, the spatial distribution of the side-chains is also compatible with those in an alpha-helix. This implies that GroEL can bind a wide range of structures, from extended beta-strands and alpha-helices to folded states, with exposed side-chains. The binding site can accommodate substrates of approximately 18 residues when in a helical or seven when in an extended conformation. The data support two activities of GroEL: the ability to act as a temporary parking spot for sticky intermediates by binding many motifs; and an unfolding activity of GroEL by binding an extended sequential conformation of the substrate.     

ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis

The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 A, reveals specific recognition of the peptide alpha-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.