E3 Binding Research Articles

Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, against the extracellular domain of the human α7-nAChR were generated through alpaca immunization with cells expressing a human α7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4β2 and α3β4. E3 acts as a slowly associating positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not precluding the desensitization of the receptor. An E3–E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with α-bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

An Aptamer That Rapidly Internalizes into Cancer Cells Utilizes the Transferrin Receptor Pathway

Strategies to direct drugs specifically to cancer cells have been increasingly explored, and significant progress has been made toward such targeted therapy. For example, drugs have been conjugated into tumor-targeting antibodies to enable delivery directly to tumor cells. Aptamers are an attractive class of molecules for this type of drug targeting as they are high-affinity/high-specificity ligands, relatively small in size, GMP manufacturable at a large-scale, amenable to chemical conjugation, and not immunogenic. Previous work from our group revealed that an aptamer selected to internalize into human prostate cancer cells, called E3, can also target a broad range of human cancers but not normal control cells. Moreover, this E3 aptamer can deliver highly cytotoxic drugs to cancer cells as Aptamer-highly Toxic Drug Conjugates (ApTDCs) and inhibit tumor growth in vivo. Here, we evaluate its targeting mechanism and report that E3 selectively internalizes into cancer cells utilizing a pathway that involves transferrin receptor 1 (TfR 1). E3 binds to recombinant human TfR 1 with high affinity and competes with transferrin (Tf) for binding to TfR1. In addition, knockdown or knockin of human TfR1 results in a decrease or increase in E3 cell binding. Here, we reported a molecular model of E3 binding to the transferrin receptor that summarizes our findings.

Nitric Oxide Mediates Direct Restriction of Pyruvate Dehydrogenase Complex via Generation of Nitroxyl During Macrophage Polarization

M1 macrophages “commit” to a metabolic state where increased aerobic glycolysis sustains fast ATP production, independently of Oxidative Phosphorylation (OXPHOS). This is associated with a “broken” mitochondrial TCA cycle and accumulation of metabolites like citrate, succinate and itaconate, important for cellular functions. We have shown that induced Nitric Oxide (NO) levels in Bone Marrow Derived Macrophages (BMDMs) from Wild Type (WT) mice activated with lipopolysaccharide and interferon gamma (LPS + IFNγ) are necessary and sufficient for the repression of OXPHOS and for metabolic reprogramming at mitochondrial Aconitase (ACO2) and Pyruvate Dehydrogenase (PDH). We hypothesize that NOdirectly targets the PDH complex via its critical cofactor lipoate, interacting with its thiol motifs. High-sensitivity proteomics on PDH complex shows that stimulated BMDM and cells exposed to NO have stalled enzymatic machinery, and this correlates with NO-associated lipoate on E2 subunit of PDH. Moreover, through native in gel activity assays we found decreased enzymatic activity of PDH-E3 and shifts in molecular weight of both PDH-E3 and E3 binding protein (E3Bp); in addition we found these positive for nitrogen specie-mediated modifications, which were not easily reversible with common reducing agents. Using chemical approaches we show that interaction of lipoate with NO can generate a reduced NO radical, nitroxyl (HNO), which can interact with thiols to form unstable intermediates that spontaneously rearrange to a sulfinamide; this represents a nearly irreversible thiol modification. Consistently, mass spectrometry analysis of recombinant protein reveals HNO-dependent post translational modification (PTM) of PDHE3. Structurally, HNO-specific modifications on E3 strongly impair homodimer formation, essential for activity. Our work is the first to demonstrate that HNO is produced physiologically in proinflammatory macrophages, and it is locally released within the interaction with lipoate moiety at the level of PDH-E2 and E3Bp, facilitating irreversible modification of distal E3. In turn this leads to inability for deacetylation of lipoate and global impairment of enzyme function. Finally, since the same E3 subunit is shared with two other key cellular metabolic enzymes, Oxoglutarate and Branched-chain alpha-keto acid Dehydrogenases (OGDH and BCKDH) we predict that NO could be responsible for orchestrating macrophage energy metabolism during inflammation also by limiting the activity of these dehydrogenases through irreversible modifications, opening the possibility for new therapeutic manipulations.

Pyruvate dehydrogenase complex deficiency: updating the clinical, metabolic and mutational landscapes in a cohort of Portuguese patients

BackgroundThe pyruvate dehydrogenase complex (PDC) catalyzes the irreversible decarboxylation of pyruvate into acetyl-CoA. PDC deficiency can be caused by alterations in any of the genes encoding its several subunits. The resulting phenotype, though very heterogeneous, mainly affects the central nervous system. The aim of this study is to describe and discuss the clinical, biochemical and genotypic information from thirteen PDC deficient patients, thus seeking to establish possible genotype–phenotype correlations.ResultsThe mutational spectrum showed that seven patients carry mutations in the PDHA1 gene encoding the E1α subunit, five patients carry mutations in the PDHX gene encoding the E3 binding protein, and the remaining patient carries mutations in the DLD gene encoding the E3 subunit. These data corroborate earlier reports describing PDHA1 mutations as the predominant cause of PDC deficiency but also reveal a notable prevalence of PDHX mutations among Portuguese patients, most of them carrying what seems to be a private mutation (p.R284X). The biochemical analyses revealed high lactate and pyruvate plasma levels whereas the lactate/pyruvate ratio was below 16; enzymatic activities, when compared to control values, indicated to be independent from the genotype and ranged from 8.5% to 30%, the latter being considered a cut-off value for primary PDC deficiency. Concerning the clinical features, all patients displayed psychomotor retardation/developmental delay, the severity of which seems to correlate with the type and localization of the mutation carried by the patient. The therapeutic options essentially include the administration of a ketogenic diet and supplementation with thiamine, although arginine aspartate intake revealed to be beneficial in some patients. Moreover, in silico analysis of the missense mutations present in this PDC deficient population allowed to envisage the molecular mechanism underlying these pathogenic variants.ConclusionThe identification of the disease-causing mutations, together with the functional and structural characterization of the mutant protein variants, allow to obtain an insight on the severity of the clinical phenotype and the selection of the most appropriate therapy.

Refinement of the clinical and mutational spectrum of UBE2A deficiency syndrome.

UBE2A deficiency, that is, intellectual disability (ID) Nascimento type (MIM 300860), is an X-linked syndrome characterized by developmental delay, moderate to severe ID, seizures, dysmorphisms, skin anomalies, and urogenital malformations. Forty affected subjects have been reported thus far, with 31 cases having intragenic UBE2A variants. Here, we report on additional eight affected subjects from seven unrelated families who were found to be hemizygous for previously unreported UBE2A missense variants (p.Glu62Lys, p.Arg95Cys, p.Thr99Ala, and p.Arg135Trp) or small in-frame deletions (p.Val81_Ala83del, and p.Asp101del). A wide phenotypic spectrum was documented in these subjects, ranging from moderate ID associated with mild dysmorphisms to severe features including congenital heart defects (CHD), severe cognitive impairment, and pineal gland tumors. Four variants affected residues (Glu62, Arg95, Thr99 and Asp101) that contribute to stabilizing the structure of the E3 binding domain. The three-residue in-frame deletion, p.Val81_Ala83del, resulted from aberrant processing of the transcript. This variant and p.Arg135Trp mapped to regions of the protein located far from the E3 binding region, and caused variably accelerated protein degradation. By reviewing available clinical information, we revise the clinical and molecular profile of the disorder and document genotype-phenotype correlations. Pineal gland cysts/tumors, CHD and hypogammaglobulinemia emerge as recurrent features.

Positive selection of cereblon modified function including its E3 ubiquitin ligase activity and binding efficiency with AMPK

Cereblon (CRBN) is a substrate receptor for an E3 ubiquitin ligase that directly binds to target proteins resulting in cellular activities, such as energy metabolism, membrane potential regulation, and transcription factor degradation. Genetic mutations in human CRBN lead to intellectual disabilities. In addition, it draws pathological attention because direct binding with immunomodulatory drugs can cure multiple myeloma (MM) and lymphocytic leukemia. To further explore the function of CRBN, we focused on its molecular evolution. Since CRBN interacts directly with its substrates and is widely conserved in vertebrates, evolutionary study to identify the selective pressure on CRBN that occur during CRBN-substrate interaction is an effective approach to search for a novel active site. Using mammalian CRBN sequences, dN/dS analysis was conducted to detect positive selection. By multiple sequence alignment we found that the residue at position 366 was under positive selection. This residue is present in the substrate-binding domain of CRBN. Most mammals harbor cysteine at position 366, whereas rodents and chiroptera have serine at this site. Subsequently, we constructed a C366S human CRBN to confirm the potential of positive selection. Auto-ubiquitination activity occurs in E3 ubiquitin ligases, including CRBN, and increased in C366S CRBN, which lead to the conclusion that E3 ubiquitin ligase activity may have changed over the course of mammalian evolution. Furthermore, binding with AMP-activated protein kinase was augmented when the substitution was present, which is supported by coevolution analysis. These results suggest that the molecular evolution of CRBN occurred through codon-based positive selection, providing a new approach to investigate CRBN function.

Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase.

The artificial WSTF PHD_EL5 RING finger was designed via “α-helical region substitution”, and its structural model for the attachment of activated ubiquitin has been demonstrated. Chemical modifications of Cys residues, the circular dichroism spectra, and substrate-independent ubiquitination assays illustrated that the WSTF PHD_EL5 RING finger has E3 activity, and it is ubiquitinated via Lys14. Homology modeling calculations revealed that the WSTF PHD_EL5 RING finger possesses a classical RING fold for specific E2–E3 binding. The docking poses of the WSTF PHD_EL5 RING finger with the UbcH5b–ubiquitin conjugate provided insight into its functional E2 interaction and development of ubiquitination at the atomic level. The structural model of the artificial WSTF PHD_EL5 RING finger proposed by the present work is useful and may help to extend the strategy of α-helical region substitution.

Activation of UbcH5c∼Ub Is the Result of a Shift in Interdomain Motions of the Conjugate Bound to U-Box E3 Ligase E4B

Post-translational modification of proteins with ubiquitin is mediated by dynamic multienzyme machinery (E1, E2, and E3). E3 ubiquitin ligases play a key role acting as both scaffolds to bring reactants together and activators to catalyze ubiquitin (Ub) transfer from E2~Ub conjugates to substrates. Our recent studies provided insights into the mechanism of the activation event; binding of an E3 to an E2~Ub conjugate was found to affect the motions of E2~Ub and allosterically stimulate Ub transfer. This proposed mechanism implies that the dynamics of the conjugate, which has been shown to occupy a wide range of E2~Ub orientations, will be altered significantly upon binding of E3. To directly assess the effect of E3 binding on E2~Ub dynamics, we undertook an in-depth comparative analysis of (15)N nuclear magnetic resonance relaxation of UbcH5c~Ub in the absence and presence of the E3 ligase, E4B. Challenges encountered in deciphering interdomain motions for this ternary complex are discussed along with the limitations of the current approaches. Notably, although a reduction in interdomain dynamics of UbcH5c~Ub is observed upon binding to E4B, Ub retains an extensive degree of flexibility. These results provide strong support for our dynamic model of a significant orientational bias of Ub toward a more closed conformation in the E3/E2~Ub complex.

RING-between-RINGs-keeping the safety on loaded guns

The ‘RING‐between‐RING’‐type E3 ubiquitin ligase HOIP acts via a novel RING/HECT‐hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non‐covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto‐inhibited. This explains why assembly of either HOIP/HOIL‐1L or HOIP/SHARPIN is required to catalyse linear chain formation.

Structure of an E3:E2∼Ub Complex Reveals an Allosteric Mechanism Shared among RING/U-box Ligases

Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here, we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 side chain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.

Pyruvate dehydrogenase deficiency caused by a new mutation of PDHX gene in two Moroccan patients

Pyruvate dehydrogenase deficiency is one of the genetic defects of mitochondrial energy metabolism. Clinical features are heterogeneous, ranging from fatal lactic acidosis in the newborn period to chronic neurodegenerative abnormalities. Most cases have mutations in the gene for the E1alpha subunit of the pyruvate dehydrogenase complex. Primary defects of the E3 binding protein component of the pyruvate dehydrogenase complex are rarier. We describe two unrelated Moroccan patients with the same new mutation c.1182 + 2T > C in the E3 binding protein gene PDHX and different clinical forms.

Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera

Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits – basic and toxic phospholipase A2 (PLA2) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 109 pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones – D12, E3, F6, D10 and G5 exhihest binding affinity for the toxic component. Clones A1, D12 and C12 recognized preferentially vipoxin's acidic component. Clones E3, G5 and H4 inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E3, G5, H4, C12, D10 and A11) inhibited direct hemolytic activity of vipoxin and its pure PLA2 subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A2 activity for the vipoxin toxicity and its effective neutralization.

The mechanism of OTUB1-mediated inhibition of ubiquitination.

SUMMARYHistones are ubiquitinated in response to DNA double strand breaks (DSB), promoting recruitment of repair proteins to chromatin1. UBC13 (UBE2N) is an ubiquitin conjugating enzyme (E2) that heterodimerizes with UEV1a2 and synthesizes K63–linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF1683. K63Ub synthesis is regulated in a noncanonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13~Ub thiolester4. Residues N-terminal to the OTU domain, which had been implicated in ubiquitin binding5, are required for binding to UBC13~Ub and inhibition of K63Ub synthesis5. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13~Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13~Ub, while at the same time disrupting interactions with UEV1a in a manner that depends upon the OTUB1 N-terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13~Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N-terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13~Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer6,7. The N-terminal helix of OTUB1 is positioned to interfere with UEV1a binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes4 in a non-catalytic manner.

Human proteome-scale structural modeling of E2-E3 interactions exploiting interface motifs.

Ubiquitination is crucial for many cellular processes such as protein degradation, DNA repair, transcription regulation, and cell signaling. Ubiquitin attachment takes place via a sequential enzymatic cascade involving ubiquitin activation (by E1 enzymes), ubiquitin conjugation (by E2 enzymes), and ubiquitin substrate tagging (by E3 enzymes). E3 ligases mediate ubiquitin transfer from E2s to substrates and as such confer substrate specificity. Although E3s can interact and function with numerous E2s, it is still unclear how they choose which E2 to use. Identifying all E2 partners of an E3 is essential for inferring the principles guiding E2 selection by an E3. Here we model the interactions of E3 and E2 proteins in a large, proteome-scale strategy based on interface structural motifs, which allows elucidation of (1) which E3s interact with which E2s in the human ubiquitination pathway and (2) how they interact with each other. Interface analysis of E2-E3 complexes reveals that loop L1 of E2s is critical for binding; the residue in the sixth position in loop L1 is widely utilized as an interface hot spot and appears indispensible for E2 interactions. Other loop L1 residues also confer specificity on the E2-E3 interactions: HECT E3s are in contact with the residue in the second position in loop L1 of E2s, but this is not the case for the RING finger type E3s. Our modeled E2-E3 complexes illuminate how slight sequence variations in E2 residues may contribute to specificity in E3 binding. These findings may be important for discovering drug candidates targeting E3s, which have been implicated in many diseases.

Membrane proteins in four acts: Function precedes structure determination

Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b 6 f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B 12 binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a “fishing pole” model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83 Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) α-Synuclein, associated with the etiology of Parkinson’s Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly α-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, α-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid–protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of α-synuclein.

Entropy-Driven Mechanism of an E3 Ligase

Ubiquitin-like modifications are macromolecular chemistry for which our understanding of the enzymatic mechanisms is lacking. Most E3 ligases in ubiquitin-like modifications do not directly participate in chemistry but are thought to confer allosteric effects; however, the nature of the allosteric effects has been elusive. Recent molecular dynamics simulations suggested that an E3 binding enhances the population of the conformational states of the E2·SUMO thioester that favor reactions. In this study, we conducted the first temperature-dependent enzyme kinetic analysis to investigate the role of an E3 on activation entropy and enthalpy. The small ubiquitin-like modifier (SUMO) E3, RanBP2, confers unusually large, favorable activation entropy to lower the activation energy of the reaction. Mutants of RanBP2, designed to alter the flexibilities of the E2·SUMO thioester, showed a direct correlation of their favorable entropic effects with their ability to restrict the conformational flexibility of the E2·SUMO thioester. While the more favorable activation entropy is consistent with the previously suggested role of E3 in conformational selection, the large positive entropy suggests a significant role of solvent in catalysis. Indeed, molecular dynamics simulations in explicit water revealed that the more stable E2·SUMO thioester upon E3 binding results in stabilization of a large number of bound water molecules. Liberating such structured water at the transition state can result in large favorable activation entropy but unfavorable activation enthalpy. The entropy-driven mechanism of the E3 is consistent with the lack of structural conservation among E3s despite their similar functions. This study also illustrates how proteins that bind both SUMO and E2 can function as E3s and how intrinsically unstructured proteins can enhance macromolecular chemistry in addition to their known advantages in protein--protein interactions.

Crystal structure of UBA2(ufd)-Ubc9: insights into E1-E2 interactions in Sumo pathways.

Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2ufd), alone and in complex with Ubc9. The overall structures of both yeast Uba2ufd and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2ufd-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2ufd-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades.

A mechanistic view of the role of E3 in sumoylation.

Sumoylation, the covalent attachment of SUMO (Small Ubiquitin-Like Modifier) to proteins, differs from other Ubl (Ubiquitin-like) pathways. In sumoylation, E2 ligase Ubc9 can function without E3 enzymes, albeit with lower reaction efficiency. Here, we study the mechanism through which E3 ligase RanBP2 triggers target recognition and catalysis by E2 Ubc9. Two mechanisms were proposed for sumoylation. While in both the first step involves Ubc9 conjugation to SUMO, the subsequent sequence of events differs: in the first E2-SUMO forms a complex with the target and E3, followed by SUMO transfer to the target. In the second, Ubc9-SUMO binds to the target and facilitates SUMO transfer without E3. Using dynamic correlations obtained from explicit solvent molecular dynamic simulations we illustrate the key roles played by allostery in both mechanisms. Pre-existence of conformational states explains the experimental observations that sumoylation can occur without E3, even though at a reduced rate. Furthermore, we propose a mechanism for enhancement of sumoylation by E3. Analysis of the conformational ensembles of the complex of E2 conjugated to SUMO illustrates that the E2 enzyme is already largely pre-organized for target binding and catalysis; E3 binding shifts the equilibrium and enhances these pre-existing populations. We further observe that E3 binding regulates allosterically the key residues in E2, Ubc9 Asp100/Lys101 E2, for the target recognition.

Identification of an unconventional E3 binding surface on the UbcH5 ∼ Ub conjugate recognized by a pathogenic bacterial E3 ligase.

Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ~ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ~ Ub(n)). Rapid generation of UbcH5 ~ Ub(n) may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.

Subunit and Catalytic Component Stoichiometries of an in Vitro Reconstituted Human Pyruvate Dehydrogenase Complex

The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772-19780).