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.     

The F‐box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen‐activated protein kinase (MAPK) pathways in Fusarium oxysporum

F‐box proteins determine substrate specificity of the ubiquitin–proteasome system. Previous work has demonstrated that the F‐box protein Fbp1, a component of the SCF^Fbp1 E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen‐activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.     

The N-end rule pathway and regulation by proteolysis

The N‐end rule relates the regulation of the in vivo half‐life of a protein to the identity of its N‐terminal residue. Degradation signals (degrons) that are targeted by the N‐end rule pathway include a set called N‐degrons. The main determinant of an N‐degron is a destabilizing N‐terminal residue of a protein. In eukaryotes, the N‐end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N‐end rule and the Arg/N‐end rule pathways. The Ac/N‐end rule pathway targets proteins containing N^α‐terminally acetylated (Nt‐acetylated) residues. The Arg/N‐end rule pathway recognizes unacetylated N‐terminal residues and involves N‐terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt‐acetylated. Thus, most proteins harbor a specific degradation signal, termed ^AcN‐degron, from the moment of their birth. Specific N‐end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N‐degrons include methionine‐aminopeptidases, caspases, calpains, Nt‐acetylases, Nt‐amidases, arginyl‐transferases, and leucyl‐transferases. Regulated degradation of specific proteins by the N‐end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights.     

Multifaceted N-Degron Recognition and Ubiquitylation by GID/CTLH E3 Ligases

N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, we discovered capacity of Gid4 and Gid10 substrate receptor subunits of yeast “GID”/human “CTLH” multiprotein E3 ligases to tightly bind a wide range of N-terminal residues whose recognition is determined in part by the downstream sequence context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues binding Gid4 or Gid10 with high affinity. Structural data reveal that conformations of flexible loops in Gid4 and Gid10 complement sequences and folds of interacting peptides. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.     

Structural basis of N‐end rule substrate recognition in Escherichia coli by the ClpAP adaptor protein ClpS

In Escherichia coli, the ClpAP protease, together with the adaptor protein ClpS, is responsible for the degradation of proteins bearing an amino‐terminal destabilizing amino acid (N‐degron). Here, we determined the three‐dimensional structures of ClpS in complex with three peptides, each having a different destabilizing residue—Leu, Phe or Trp—at its N terminus. All peptides, regardless of the identity of their N‐terminal residue, are bound in a surface pocket on ClpS in a stereo‐specific manner. Several highly conserved residues in this binding pocket interact directly with the backbone of the N‐degron peptide and hence are crucial for the binding of all N‐degrons. By contrast, two hydrophobic residues define the volume of the binding pocket and influence the specificity of ClpS. Taken together, our data suggest that ClpS has been optimized for the binding and delivery of N‐degrons containing an N‐terminal Phe or Leu.     

Does N‐Terminal Protein Acetylation Lead to Protein Degradation?

The N‐end rule denotes the relationship between the identity of the amino‐terminal residue of a protein and its in vivo half‐life. Since its discovery in 1986, the N‐end rule has generally been described by a defined set of rules for determining whether an amino‐terminal residue is stabilizing or not. However, recent studies are revealing that this N‐end rule (or N‐degron concept) is less straightforward than previously appreciated. For instance, it is unveiled that N‐terminal acetylation of N‐terminal residues may create a degradation signal (Ac‐degron) that promotes the degradation of target proteins. A recent high‐throughput dissection of degrons in yeast proteins amino termini intriguingly suggested that the hydrophobicity of amino‐terminal residues—but not the N‐terminal acetylation status—may be the indispensable feature of amino‐terminal degrons. Herein, these recent advances in N‐terminal acetylation and the complexity of N‐terminal degradation signals in the context of the N‐degron pathway are analyzed.     

Structure of a putative ClpS N‐end rule adaptor protein from the malaria pathogen Plasmodium falciparum

The N‐end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP‐dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N‐terminal amino acids (N‐degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N‐end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N‐degrons. However, while the N‐degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal‐ClpS binds and discriminates peptides mimicking bona fide N‐end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal‐ClpS localizes to this plastid‐like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti‐malarial drugs aimed at disrupting parasite‐specific protein quality control pathways.     

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.     

Binding to serine 65‐phosphorylated ubiquitin primes Parkin for optimal PINK1‐dependent phosphorylation and activation

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser⁶⁵)—which lies within its ubiquitin‐like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser⁶⁵. How Ser⁶⁵‐phosphorylated ubiquitin (ubiquitin^{Phospho‐Ser65}) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin^{Phospho‐Ser65} binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser⁶⁵ by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site‐directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin^{Phospho‐Ser65}, thereby promoting Parkin Ser⁶⁵ phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser⁶⁵ phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin^{Phospho‐Ser65} to Parkin disrupts the interaction between the Ubl domain and C‐terminal region, thereby increasing the accessibility of Parkin Ser⁶⁵. Finally, purified Parkin maximally phosphorylated at Ser⁶⁵ in vitro cannot be further activated by the addition of ubiquitin^{Phospho‐Ser65}. Our results thus suggest that a major role of ubiquitin^{Phospho‐Ser65} is to promote PINK1‐mediated phosphorylation of Parkin at Ser⁶⁵, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho‐Ser⁶⁵‐binding pocket on the surface of Parkin that is critical for the ubiquitin^{Phospho‐Ser65} interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin^{Phospho‐Ser65}, which could aid in the development of Parkin activators that mimic the effect of ubiquitin^{Phospho‐Ser65}.     

Targeted Disruption of an EH‐domain Protein Endocytic Complex,Pan1‐End3

Pan1 is a multi‐domain scaffold that enables dynamic interactions with both structural and regulatory components of the endocytic pathway. Pan1 is composed of Eps15 Homology (EH) domains which interact with adaptor proteins, a central region that is responsible for its oligomerization and C‐terminal binding sites for Arp2/3, F‐actin, and type‐I myosin motors. In this study, we have characterized the binding sites between Pan1 and its constitutive binding partner End3, another EH domain containing endocytic protein. The C‐terminal End3 Repeats of End3 associate with the N‐terminal part of Pan1's central coiled‐coil region. These repeats appear to act independently of one another as tandem, redundant binding sites for Pan1. The end3‐1 allele was sequenced, and corresponds to a C‐terminal truncation lacking the End3 Repeats. Mutations of the End3 Repeats highlight that those residues which are identical between these repeats serve as contact sites for the interaction with Pan1.      

Structural Basis of Selective Ubiquitination of TRF1 by SCFFbx4

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Highlights? SCF ubiquitin ligase subunit Fbx4 binds TRF1 using an atypical small GTPase fold ? Fbx4 recognizes a globular domain of TRF1, not just short phosphorylated degrons ? Telomere shelterin component TIN2 competes with Fbx4 for TRF1 binding ? TIN2 and SCF^Fbx4 thus control TRF1 ubiquitination and degradation     

Cullin neddylation and substrate‐adaptors counteract SCF inhibition by the CAND1‐like protein Lag2 in Saccharomyces cerevisiae

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 β‐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.     

Nrdp1S,short variant of Nrdp1, inhibits human glioma progression by increasing Nrdp1‐mediated ErbB3 ubiquitination and degradation

The ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp1) is involved in the induction of apoptosis and suppression of tumour formation. We previously showed that it was expressed at lower levels in human glioma tissues compared with normal brain tissues. However, the mechanism underlying this is unclear. Here, we reported that a novel short variant (Nrdp1S), lacking 71 amino acids at the N‐terminal, was expressed in normal human brain tissue, but absent from glioma tissues. Similar to Nrdp1, Nrdp1S could be degraded by the proteasomal pathway, but exhibited an even longer half‐life than Nrdp1. Nrdp1S was also shown to form a heterodimer with Nrdp1, which increased its stability, thereby augmenting the Nrdp1‐mediated ubiquitination and degradation of ErbB3. EdU incorporation, MTT assay and in vitro colony formation demonstrated that Nrdp1S significantly inhibited the cell tumourigenicity. These results together suggest that Nrdp1S is a tumour suppressor that which potentiates the Nrdp1‐mediated ubiquitination and degradation of ErbB3. An Nrdp1S deficiency may also be an important factor in the loss of Nrdp1.     

Structure of galactarate dehydratase,a new fold in an enolase involved in bacterial fitness after antibiotic treatment

Galactarate dehydratase (GarD) is the first enzyme in the galactarate/glucarate pathway and catalyzes the dehydration of galactarate to 3‐keto‐5‐dehydroxygalactarate. This protein is known to increase colonization fitness of intestinal pathogens in antibiotic‐treated mice and to promote bacterial survival during stress. The galactarate/glucarate pathway is widespread in bacteria, but not in humans, and thus could be a target to develop new inhibitors for use in combination therapy to combat antibiotic resistance. The structure of almost all the enzymes of the galactarate/glucarate pathway were solved previously, except for GarD, for which only the structure of the N‐terminal domain was determined previously. Herein, we report the first crystal structure of full‐length GarD solved using a seleno‐methoionine derivative revealing a new protein fold. The protein consists of three domains, each presenting a novel twist as compared to their distant homologs. GarD in the crystal structure forms dimers and each monomer consists of three domains. The N‐terminal domain is comprised of a β‐clip fold, connected to the second domain by a long unstructured linker. The second domain serves as a dimerization interface between two monomers. The C‐terminal domain forms an unusual variant of a Rossmann fold with a crossover and is built around a seven‐stranded parallel β‐sheet supported by nine α‐helices. A metal binding site in the C‐terminal domain is occupied by Ca²⁺. The activity of GarD was corroborated by the production of 5‐keto‐4‐deoxy‐D‐glucarate under reducing conditions and in the presence of iron. Thus, GarD is an unusual enolase with a novel protein fold never previously seen in this class of enzymes.     

Molecular dynamic studies on the impact of mutations on the structure,stability, and N‐terminal orientation of annexin A1: Implications for membrane aggregation

Multiple MD simulations were performed for the full‐length wild‐type A1, the full length A1 mutations S27E and S27A, as well as the N‐terminal peptide (AMVSEFLKQAWFIDNEEQEYIKTVKG S ²⁷ KGGPGSAVSPYPTFN) of wild‐type A1 and mutations S27E and S27A. The MD simulation trajectories of about 350 ns were generated and analyzed to examine the changes of core domain calcium binding affinity, core domain and N‐terminal domain structures, and N‐terminal domain orientation. Our results indicated that S27A and S27E mutations caused little changes on the calcium‐binding affinity of the core domain of A1. However, the S27A mutation made the N‐terminal domain of A1 less helical, and made the N‐terminal domain migrate faster toward the core domain; these impacts on A1 are beneficial to the membrane aggregation process. On the contrary, the S27E mutation made the N‐terminal domain of A1 more stable, and hindered the migration to the core domain; these changes on A1 are antagonistic for the membrane aggregation process. Our results using MD simulations provide an atomistic explanation for experimental observations that the S27E mutant showed a higher calcium concentration requirement and lower maximal extent of aggregation, while the wild‐type and two mutants S27E and S27A required identical calcium concentrations for liposome binding. Proteins 2014; 82:3327–3334. © 2014 Wiley Periodicals, Inc.     

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms

The tumor‐suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N‐terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain‐containing proteins. RING domains bind and activate E2 ubiquitin‐conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer‐associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.     

The Hsp70 chaperone Ssa1 is essential for catabolite induced degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme of gluconeogenesis. In the yeast Saccharomyces cerevisiae, it is only expressed when cells are grown in medium with nonfermentable carbon sources. Addition of glucose to cells leads to inactivation of FBPase and degradation via the ubiquitin-proteasome system. Polyubiquitination of FBPase is carried out by the Gid complex, a multi-subunit ubiquitin ligase. Using tandem affinity purification and subsequent mass spectrometry we identified the Hsp70 chaperone Ssa1 as a novel interaction partner of FBPase. Studies with the temperature-sensitive mutant ssa1-45^ts showed that Ssa1 is essential for polyubiquitination of FBPase by the Gid complex. Moreover, we show that degradation of an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is also affected in ssa1-45^ts cells demonstrating that Ssa1 plays a general role in elimination of gluconeogenic enzymes.     

The Cdc48-Ufd1-Npl4 complex is central in ubiquitin-proteasome triggered catabolite degradation of fructose-1,6-bisphosphatase

The switch from gluconeogenesis to glycolysis in yeast has been shown to require ubiquitin-proteasome dependent elimination of the key enzyme fructose-1,6-bisphosphatase (FBPase). Prior to proteasomal degradation, polyubiquitination of the enzyme occurs via the ubiquitin-conjugating enzymes Ubc1, Ubc4, Ubc5 and Ubc8 in conjunction with a novel multi-subunit ubiquitin ligase, the Gid complex. As an additional machinery required for the catabolite degradation process, we identified the trimeric Cdc48^Ufd1-Npl4 complex and the ubiquitin receptors Dsk2 and Rad23. We show that this machinery acts between polyubiquitination of FBPase and its degradation by the proteasome.