Binding Of E3 Research Articles

An original potentiating mechanism revealed by the cryo-EM structures of the human α7 nicotinic receptor in complex with nanobodies

The human α7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest in designing ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of α7 ligands, we recently generated two nanobodies named E3 and C4, acting as positive allosteric modulator and silent allosteric ligand, respectively. Here, we solved the cryo-electron microscopy structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an agonist-bound conformation of the extracellular domain in the absence of an orthosteric agonist, and mutational analysis shows a key contribution of an N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation that opens new avenues for drug design.

Lecithin coating as universal stabilization and functionalization strategy for nanosized drug carriers to overcome the blood-brain barrier.

Lecithin coated cholesteryl oleate (ChOl) based nanoparticles (NPs) imitating natural lipoproteins represent a new and promising drug carrier strategy to cross the blood-brain barrier (BBB). In such systems lecithin serves as stabilizing as well as functionalizing agent and enables the adsorptive binding of apolipoprotein E3 (ApoE) as potential drug targeting ligand. The present work is focused on the effect of size reduction on the lecithin coating and ApoE binding. Furthermore, the transferability of this lecithin coating strategy to other NP cores, namely polylactic-co-glycolic acid (PLGA) and polylactic acid (PLA), is investigated in order to provide a universal strategy for a wide range of cores to overcome the BBB. The ChOl NPs' size was successfully reduced from 100nm to 70nm. Varying the core size of ChOl NPs illustrated, that the at least needed lecithin amount for sufficient stabilization could be calculated surface area dependently. However, the size reduction led to reduced dye loading per NP and increased ApoE need per NP mass. These effects turned out as huge disadvantages of smaller NPs by weakening the observed ApoE mediated effects. Nevertheless, the extended understanding of the lecithin coating could be used to transfer the concept to other core materials. PLGA and PLA NPs were investigated as alternative core materials for lecithin coating. PLGA was found to be unsuitable, whereas in the case of PLA sufficient stabilization and 100% adsorptive binding efficiency to ApoE could be achieved. The ApoE mediated effects of transcytosis at an in vitro BBB model by bypassing lysosomes were reproduced in even stronger quantities than with a ChOl core, proving lecithin coating as transferable strategy to disguise various NPs with a certain lipophilicity as lipoproteins.

Mechanism of Lipid Binding of Human Apolipoprotein E3 by Hydrogen/Deuterium Exchange/Mass Spectrometry and Fluorescence Polarization.

Human apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in maintaining plasma cholesterol/triglyceride homeostasis. The C-terminal (CT) domain of apoE3 (residues 201-299) is composed of amphipathic α-helices C1: W210-S223, C2: V236-E266, and C3: D271-W276, which play a dominant role in mediating high-affinity lipid binding. The objective is to understand the accessibility of the CT domain at the sub-domain level and the mechanistic details regarding lipid-binding interaction. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX/MS) of recombinant wild type (WT) apoE(201-299), chemical-induced unfolding monitored as changes in fluorescence polarization (FP) of labeled apoE(201-299) bearing a probe at specified sites, and lipid binding studies were carried out. HDX/MS revealed that residues towards the C-terminal end of the domain display significantly lower %D uptake compared to those towards the center, suggesting extensive protein-protein interaction in this segment. Functional assays showed that locking apoE(201-299) in an inter-molecular disulfide-bonded state at position 209, 223, 255, or 277 significantly decreases its ability to interact with lipids, especially when tethered towards the ends; this could be restored by reduction. Unfolding studies indicate that the C-terminal end offers less resistance to unfolding compared to the central portion of the domain. Taken together, our data suggest that two dimers of CT domain are juxtaposed around helix C3 leading to apoE3 tetramerization, and that dissociation to monomeric units is a required step in lipid binding, with helix C3 likely seeking stability via lipid interaction prior to helices C1 or C2.

P130Cas scaffold protein regulates ErbB2 stability by altering breast cancer cell sensitivity to autophagy.

Overexpression of the ErbB2/HER2 receptor tyrosine kinase occurs in up to 20% of human breast cancers and correlates with aggressive disease. Several efficacious targeted therapies, including antibodies and kinase inhibitors, have been developed but the occurring of resistance to these agents is often observed. New therapeutic agents targeting the endocytic recycling and intracellular trafficking of membrane in tumor cells overexpressing ErbB2 are actually in clinical development. Nevertheless the mechanisms underlying ErbB2 downregulation are still obscure. We have previously demonstrated that the overexpression of the p130Cas adaptor protein in ErbB2 positive breast cancer, promotes tumor aggressiveness and progression. Here we demonstrate that lowering p130Cas expression in breast cancer cells is sufficient to induce ErbB2 degradation by autophagy. Conversely, p130Cas overexpression protects ErbB2 from degradation by autophagy. Furthermore, this autophagy-dependent preferential degradation of ErbB2 in absence of p130Cas is due to an increased ErbB2 ubiquitination. Indeed, the overexpression of p130Cas impairs ErbB2 ubiquitination by inhibiting the binding of Cbl and CHIP E3 ligases to ErbB2. Finally, our results indicate that p130Cas-dependent ErbB2 protection from degradation by autophagy may alter the sensitivity to the humanized monoclonal antibody trastuzumab. Consistently, in human ErbB2 positive breast cancers that develop resistance to trastuzumab, p130Cas expression is significantly increased suggesting that elevated levels of p130Cas can be involved in trastuzumab resistance.

The Role of E3 in pH Protection during Alphavirus Assembly and Exit

Alphaviruses are small enveloped viruses whose surface is covered by spikes composed of trimers of E2/E1 glycoprotein heterodimers. During virus entry, the E2/E1 dimer dissociates within the acidic endosomal environment, freeing the E1 protein to mediate fusion of the viral and endosome membranes. E2 is synthesized as a precursor, p62, which is cleaved by furin in the late secretory pathway to produce mature E2 and a small peripheral glycoprotein, E3. The immature p62/E1 dimer is acid resistant, but since p62 is cleaved before exit from the acidic secretory pathway, low pH-dependent binding of E3 to the spike complex is believed to prevent premature fusion. Based on analysis of the structure of the Chikungunya virus E3/E2/E1 complex, we hypothesized that interactions of E3 residues Y47 and Y48 with E2 are important in this binding. We then directly tested the in vivo role of E3 in pH protection by alanine substitutions of E3 Y47 and Y48 (Y47/48A) in Semliki Forest virus. The mutant was nonviable and was blocked in E1 transport to the plasma membrane and virus production. Although the Y47/48A mutant initially formed the p62/E1 heterodimer, the dimer dissociated during transport through the secretory pathway. Neutralization of the pH in the secretory pathway successfully rescued dimer association, E1 transport, and infectious particle production. Further mutagenesis identified the critical contact as the cation-π interaction of E3 Y47 with E2. Thus, E3 mediates pH protection of E1 during virus biogenesis via interactions strongly dependent on Y47 at the E3-E2 interface.

Binding and Repressive Activities of Apolipoprotein E3 and E4 Isoforms on the Human ApoD Promoter

Apolipoprotein D (ApoD) gene expression is increased in several neurological disorders such as Alzheimer’s disease (AD) and multiple sclerosis. We previously showed that transgenic mice that overexpress human ApoD show a better resistance against paraquat or OC43 coronavirus-induced neurodegeneration. Here, we identified several nuclear factors from the cortex of control and OC43-infected mice which bind a fragment of the proximal ApoD promoter in vitro. Of interest, we detected apolipoprotein E (ApoE). Human ApoE consists of three isoforms (E2, E3, and E4) with the E4 and E2 alleles representing a greater and a lower risk for developping AD, respectively. Our results show that ApoE is located in the nucleus and on the ApoD promoter in human hepatic and glioblastoma cells lines. Furthermore, overexpression of ApoE3 and ApoE4 isoforms but not ApoE2 significantly inhibited the ApoD promoter activity in U87 cells (E3/E3 genotype) cultured under normal or different stress conditions while ApoE knock-down by siRNA had a converse effect. Consistent with these results, we also demonstrated by ChIP assay that E3 and E4 isoforms, but not E2, bind the ApoD promoter. Moreover, using the Allen Brain Atlas in situ hybridization database, we observed an inverse correlation between ApoD and ApoE mRNA expression during development and in several regions of the mouse brain, notably in the cortex, hippocampus, plexus choroid, and cerebellum. This negative correlation was also observed for cortex layers IV–VI based on a new Transcriptomic Atlas of the Mouse Neocortical Layers. These findings reveal a new function for ApoE by regulating ApoD gene expression.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-013-8456-0) contains supplementary material, which is available to authorized users.

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.

How Dihydrolipoamide Dehydrogenase-binding Protein Binds Dihydrolipoamide Dehydrogenase in the Human Pyruvate Dehydrogenase Complex

The dihydrolipoamide dehydrogenase-binding protein (E3BP) and the dihydrolipoamide acetyltransferase (E2) component enzyme form the structural core of the human pyruvate dehydrogenase complex by providing the binding sites for two other component proteins, dihydrolipoamide dehydrogenase (E3) and pyruvate dehydrogenase (E1), as well as pyruvate dehydrogenase kinases and phosphatases. Despite a high similarity between the primary structures of E3BP and E2, the E3-binding domain of human E3BP is highly specific to human E3, whereas the E1-binding domain of human E2 is highly specific to human E1. In this study, we characterized binding of human E3 to the E3-binding domain of E3BP by x-ray crystallography at 2.6-angstroms resolution, and we used this structural information to interpret the specificity for selective binding. Two subunits of E3 form a single recognition site for the E3-binding domain of E3BP through their hydrophobic interface. The hydrophobic residues Pro133, Pro154, and Ile157 in the E3-binding domain of E3BP insert themselves into the surface of both E3 polypeptide chains. Numerous ionic and hydrogen bonds between the residues of three interacting polypeptide chains adjacent to the central hydrophobic patch add to the stability of the subcomplex. The specificity of pairing for human E3BP with E3 is interpreted from its subcomplex structure to be most likely due to conformational rigidity of the binding fragment of the E3-binding domain of E3BP and its exquisite amino acid match with the E3 target interface.

Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein-RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis.

Association and dissociation kinetics of colicin E3 and immunity protein 3: convergence of theory and experiment.

The rapid binding of cytotoxic colicin E3 by its cognate immunity protein Im3 is essential in safeguarding the producing cell. The X-ray structure of the E3/Im3 complex shows that the Im3 molecule interfaces with both the C-terminal ribonuclease (RNase) domain and the N-terminal translocation domain of E3. The association and dissociation rates of the RNase domain and Im3 show drastically different sensitivities to ionic strength, as previously rationalized for electrostatically enhanced diffusion-limited protein-protein associations. Relative to binding to the RNase domain, binding to full-length E3 shows a comparable association rate but a significantly lower dissociation rate. This outcome is just what was anticipated by a theory for the binding of two linked domains to a protein. The E3/Im3 system thus provides a powerful paradigm for the interplay of theory and experiment.

Role of dihydrolipoyl dehydrogenase (E3) and a novel E3-binding protein in the NADH sensitivity of the pyruvate dehydrogenase complex from anaerobic mitochondria of the parasitic nematode, Ascaris suum.

The pyruvate dehydrogenase complex (PDC) plays changing roles during the aerobic-anaerobic transition in the life cycle of the parasitic nematode, Ascaris suum. However, the dihydrolipoyl dehydrogenase (E3) subunit appears to be identical in all stages, despite the fact that the PDC is less sensitive to NADH inhibition in anaerobic muscle. Therefore, we have cloned cDNAs encoding E3 and a novel anaerobic-specific E3-binding protein (E3BP) that lacks the terminal lipoyl domain found in E3BPs from yeast and mammals, and functionally expressed E3 and E3 mutants designed to have decreased dimer stability on the assumption that the binding of E3 to an anaerobic-specific E3BP might stabilize the E3 dimer interface and decrease E3 sensitivity to NADH inhibition. As predicted, the mutants exhibited decreased thermal stability, increased sensitivity to NADH and the binding of E3(Y18F) to the E3-depleted core of the pig heart PDC increased E3 activity and decreased E3 sensitivity to NADH inhibition. However, although the free A. suum E3 was less sensitive to NADH inhibition than the pig heart E3, both E3s were significantly more sensitive to NADH inhibition when assayed with dihydrolipoamide than their corresponding PDCs assayed with pyruvate. More importantly, the binding of rE3 to its core complex had little effect on its apparent K(m) for NAD(+), K(i) for NADH inhibition, or the NADH/NAD(+) ratio yielding 50% inhibition. These data suggest that although binding to the core stabilizes the E3 dimer interface, it does not play a significant role in reducing the sensitivity of the A. suum PDC to NADH inhibition during anaerobiosis.

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.

Influence of Apolipoprotein E3 on the Production of Paired Helical Filament in Guamanian Parkinsonism‐dementia Complex/Amyotrophic Lateral Sclerosis and Alzheimer's Disease

Background: Neuropathologically, Guamanian parkinsonism‐dementia complex (PDC) and amyotrophic lateral sclerosis (ALS) are characterized by neuronal loss and neurofibrillary tangles (NFTs). The NFTs are principally composed of paired helical filaments (PHFs) consisting of hyperphosphorylated tau and have been shown to be histopathologically similar to those in Alzheimer's disease (AD). Recent biochemical studies have suggested that the binding of apolipoprotein E3 (ApoE3) to tau may interfere with the hyperphosphorylation of tau, thus resulting in neuroprotective effects. Methods: In order to clarify the influence of ApoE3 on the production of PHFs in various pathological conditions, the ɛ3 allele frequency in patients with Guamanian PDC and ALS, as well as AD, was examined. Results: The results obtained in this study showed no significant differences between these affected groups and each of the controls in terms of the frequency of the ApoE3 allele. Conclusion: Although ApoE3 has been implicated in the production of PHFs, the present study suggests that ApoE3 may not have a strong inhibitory effect on the production of NFTs in these affected patients.

Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus.

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over-expressed in Escherichia coli. Titrations of the icosahedral (60-mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, alpha2beta2) and dihydrolipoyl dehydrogenase (E3, alpha2) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3, and displacement of E1 or E3 from E1-E2 or E3-E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active-site coupling.

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro.

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.

The interaction between lipoamide dehydrogenase and the peripheral-component-binding domain from the Azotobacter vinelandii pyruvate dehydrogenase complex.

The sensitivity of lipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase E3) from Azotobacter vinelandii to inhibition by NADH requires measurement of the activity in the initial phase of the reaction. Stopped-flow turnover experiments show that kcat is 830 s-1 compared with 420 s-1 found in standard steady-state experiments. Mutations at the si-side of the flavin prosthetic group that cause severe inhibition by NADH were studied. Tyr16 was replaced by phenylalanine and serine, which causes the loss of two intersubunit H-bonds. [F16]E3 shows only 5.7% of wild-type activity in the standard assay procedure, but analyzed by stopped-flow the activity is 70% of the wild-type enzyme. The NADH-->Cl2Ind (dichloroindophenol) activity was normal or slightly increased. The inhibition by NADH is competitive with respect to NAD+, Ki = 50 microM. Spectral analysis show that electrons readily pass over from the disulfide to the FAD, indicating an increase in the redox potential of the flavin. It is concluded that subunit interaction plays an important role in the protection of the enzyme against over-reduction by decreasing the redox potential of the flavin. The interaction of wild-type or mutant enzymes with the core component of the pyruvate (E2p) or oxoglutarate (E2o) dehydrogenase multienzyme complex relieves the inhibition to a large extent. In the mutant enzymes, the mechanism of inhibition changes from competitive to the mixed-type inhibition observed for the wild-type enzyme. The stabilizing effect of E2 on [F16]E3 was used as an assay to analyze the stoichiometry of interaction of E3 with E2p as well as E2o. 1 mol E2p monomer was sufficient to saturate 1 mol E3 dimer with a Kd of about 1 nM. Similarly, 1 mol E2o saturated the E3 dimer with a Kd of 30 nM. From these experiments it is concluded that the E3-binding domain of E2 interacts with the subunit interface of E3 near the dyad axis, thus preventing sterically the interaction with a second molecule of the binding domain. This mode of interaction, which causes asymmetry in the complex, explains the stabilization against over-reduction by tightening the subunit interaction. Subgene cloning of the E2p component of the pyruvate dehydrogenase complex is described in order to obtain a complex between the lipoamide dehydrogenase component (E3) and the binding domain of E2p. A unique restriction site in the DNA encoding the flexible linker between the third lipoyl domain and the binding domain combined with timed digestion with exonuclease Bal31 was used to create a set of deletion mutants in the N-terminal region of the binding-catalytic didomain, fused to six N-terminal amino acids from beta-galactosidase. The expressed proteins, selected for E2p activity, were analyzed for binding of E3 and E1p. The shortest fusion protein containing a functional binding domain was expressed and purified. [F16]E3 was combined with this fusion protein in a stoichiometric ratio and the resulting complex was subjected to limited proteolysis to remove the catalytic domain. The resulting [F16]E3-binding domain preparation was purified to homogeneity.

Dissociation and Reassembly of the Dihydrolipoyl Transacetylase Component of the Bovine Heart Pyruvate Dehydrogenase Complex

The catalytic activity and the state of aggregation of the dihydrolipoyl transacetylase–lipoamide dehydrogenase binding protein (E2–E3BP) subcomplex of the bovine heart pyruvate dehydrogenase multienzyme complex were investigated. Treatment of E2–E3BP with the chaotropic salts GndnCl or KSCN led to a rapid decrease in transacetylase activity which was accompanied by a loss of the native quaternary structure, as indicated by changes in the sedimentation properties of the E2–E3BP subcomplex. Reassembly or refolding of dissociated E2–E3BP was achieved for the GndnCl-treated subcomplex using a defined protocol. This reassembly procedure effectively excluded all E3BP from the reassembled oligomeric transacetylase. The reassembled oligomeric E2, free of E3BP, was unable to reconstitute the overall activity of the complex following incubation with pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3). In binding studies using radiolabeled components it was demonstrated that the reassembled transacetylase, while retaining its capacity for reductive acetylation and its ability to bind E1, lost its ability to bind E3. The evidence presented in this study indicates that the strong association of E3BP with E2facilitates the binding of E3, the lipoamide dehydrogenase component, and therefore may have an important role in the assembly and ultimately the catalytic activity of the pyruvate dehydrogenase multienzyme complex.

Binding properties of the components of branched-chain 2-oxo acid dehydrogenase complex on ELISA.

Binding characteristics among three catalytic components of rat liver branched-chain 2-oxo acid dehydrogenase complex (BCKADH) were investigated by ELISA. Dihydrolipoamide dehydrogenase (E3) was bound to solid-phase dihydrolipoamide acyltransferase (E2). The binding curve was hyperbolic giving a calculated half-maximal binding concentration of 167 ng/ml for E3. Specificity of the binding of E3 to E2 was certified by a competition experiment measuring binding of biotin-labeled E3 in the presence of unlabeled E3. The decarboxylase component (E1), which is the other catalytic component of the complex, prevented the E3 binding to E2. The specific binding between E2 and E3 was verified in the opposite direction with immobilized E3. E2 also bound to solid-phase E1 in a specific manner, and addition of E3 prevented the E2 binding to E1. No binding between E1 and E3 was observed. Thus, E1 and E3 prevented each other's binding to E2, suggesting that E1 and E3 may recognize overlapped binding sites on the E2 polypeptide or that they may, at least in part, sterically interact with each other on their binding to E2. The reconstitution experiment of the complex also supported such a mutually exclusive binding of E1 and E3 to the E2 core.

Structure/function relationships in the pyruvate dehydrogenase complex form Azotobacter vinelandii

The role of the hinge region between the binding domain and the catalytic domain in dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii was addressed by deletion mutagenesis. Mutated dihydrolipoyl transacetylase proteins were constructed with a deletion of 11 amino acids in the hinge region between the binding domain and the N-terminal part of the catalytic domain of E2p [E2p(pAPE1)] and with a further deletion of 9 amino acids into the N-terminal sequence protruding from the globular structure of the catalytic domain [E2p(pAPE2)] and found to take part in the intratrimer interaction. Both proteins behaved as wild-type E2p with respect to catalytic activity and quaternary structure. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) with the mutated E2p proteins was studied. E2p(pAPE1) assembles to a trimeric pyruvate dehydrogenase complex (PDC) with 15% decreased complex activity. No difference in affinity towards the peripheral components was detected. Upon binding of E3, E2p(pAPE2) dissociates into trimers and monomers. At saturation, two dimers of E3 were bound/E2p monomer instead of one dimer/E2p chain in trimeric wild-type E2p or E2p (pAPE1). The monomeric E2p species was catalytically inactive. Upon binding of excess E1p, some monomer formation of the E2p mutant took place. E1p however can prevent monomerization by E3. It is concluded that E1p is bound between two different E2p chains in the trimer. The substrates CoA and acetyl-CoA also prevent monomerization because they are bound by amino acid residues of two different E2p chains. In the presence of CoA no difference in affinity with respect to E1p and E3 binding was observed. CoA (and acetylCoA) also prevent dissociation of the 24-subunit core structure of wild-type E2p when added before addition of E1p or E3. Therefore, it seems likely that in vivo A. vinelandii PDC is based on a 24-subunit E2p core, like Escherichia coli PDC. A functional difference between complexes based on a trimer or a 24-subunit core has not been observed. A role of the hinge region as a spacer to allow binding of E1p or E3 seems unlikely. The results are discussed on the basis of the three-dimensional structure of the catalytic domain.

The pyruvate dehydrogenase complex from the parasitic nematode Ascaris suum: Novel subunit composition and domain structure of the dihydrolipoyl transacetylase component

The pyruvate dehydrogenase complex (PDC) from muscle of the adult parasitic nematode Ascaris suum plays a unique role in its anaerobic mitochondrial metabolism. Resolution of the intact complex in high salt dissociates the pyruvate dehydrogenase subunit but leaves the dihydrolipoyl dehydrogenase subunit (E3) and two other proteins with apparent M r s of 45 and 43 kDa bound to the dihydrolipoyl transacetylase (E2) core. These proteins are not observable on Coomassie brilliant blue-stained gels of other eukaryotic PDCs, but the 45-kDa protein is similar in apparent M r , p I, and sensitivity to trypsin to the K b subunit of the bovine kidney PDH a kinase. Acetylation of the ascarid PDC with [2- 14C]pyruvate under conditions designed to maximize the incorporation of label into protein yielded only a single radiolabeled subunit, E2. These results confirm earlier reports that the ascarid PDC lacks protein X, an integral component recently identified in other eukaryotic PDCs. About 1.6 to 1.8 mol of 14C was incorporated/mole of E2, suggesting that the ascarid E2 contained two lipoylbearing domains. Domain mapping of the 14C-acetylated ascarid E2 by limited tryptic digestion identified two lipoyl-bearing fragments with apparent M r s of 50 and 34 kDa and two core fragments with apparent M r s of 46 and 30 kDa. The ascarid E2 domain structure appears to be similar to that of other E2s. However, it appears that the subunit-binding domain (E2 B) of the ascarid E2 may be significantly larger or be flanked by larger than normal interdomain regions. An enlarged E2 B domain may be necessary to accomodate the additional binding of E3 to the E2 subunit in the ascarid complex, in the absence of protein X.