Dimerization of a 5-kDa domain defines the architecture of the 5-MDa gammaproteobacterial pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E23), we show that two of the three PSBDs in the E23 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E23. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.

Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion.

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.

The phytochemical hyperforin triggers thermogenesis in adipose tissue via a Dlat-AMPK signaling axis to curb obesity.

Stimulation of adipose tissue thermogenesis is regarded as a promising avenue in the treatment of obesity. However, pharmacologic engagement of this process has proven difficult. Using the Connectivity Map (CMap) approach, we identified the phytochemical hyperforin (HPF) as an anti-obesity agent. We found that HPF efficiently promoted thermogenesis by stimulating AMPK and PGC-1α via a Ucp1-dependent pathway. Using LiP-SMap (limited proteolysis-mass spectrometry) combined with a microscale thermophoresis assay and molecular docking analysis, we confirmed dihydrolipoamide S-acetyltransferase (Dlat) as a direct molecular target of HPF. Ablation of Dlat significantly attenuated HPF-mediated adipose tissue browning both in vitro and in vivo. Furthermore, genome-wide association study analysis indicated that a variation in DLAT is significantly associated with obesity in humans. These findings suggest that HPF is a promising lead compound in the pursuit of a pharmacological approach to promote energy expenditure in the treatment of obesity.

Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase.

The pyruvate dehydrogenase multienzyme complex (PDHc) connects glycolysis to the tricarboxylic acid cycle by producing acetyl-CoA via the decarboxylation of pyruvate. Because of its pivotal role in glucose metabolism, this complex is closely regulated in mammals by reversible phosphorylation, the modulation of which is of interest in treating cancer, diabetes, and obesity. Mutations such as that leading to the αV138M variant in pyruvate dehydrogenase, the pyruvate-decarboxylating PDHc E1 component, can result in PDHc deficiency, an inborn error of metabolism that results in an array of symptoms such as lactic acidosis, progressive cognitive and neuromuscular deficits, and even death in infancy or childhood. Here we present an analysis of two X-ray crystal structures at 2.7-Å resolution, the first of the disease-associated human αV138M E1 variant and the second of human wildtype (WT) E1 with a bound adduct of its coenzyme thiamin diphosphate and the substrate analogue acetylphosphinate. The structures provide support for the role of regulatory loop disorder in E1 inactivation, and the αV138M variant structure also reveals that altered coenzyme binding can result in such disorder even in the absence of phosphorylation. Specifically, both E1 phosphorylation at αSer-264 and the αV138M substitution result in disordered loops that are not optimally oriented or available to efficiently bind the lipoyl domain of PDHc E2. Combined with an analysis of αV138M activity, these results underscore the general connection between regulatory loop disorder and loss of E1 catalytic efficiency.

Atomic Structure of the E2 Inner Core of Human Pyruvate Dehydrogenase Complex.

Pyruvate dehydrogenase complex (PDC) is a large multienzyme complex that catalyzes the irreversible conversion of pyruvate to acetyl-coenzyme A with reduction of NAD+. Distinctive from PDCs in lower forms of life, in mammalian PDC, dihydrolipoyl acetyltransferase (E2; E2p in PDC) and dihydrolipoamide dehydrogenase binding protein (E3BP) combine to form a complex that plays a central role in the organization, regulation, and integration of catalytic reactions of PDC. However, the atomic structure and organization of the mammalian E2p/E3BP heterocomplex are unknown. Here, we report the structure of the recombinant dodecahedral core formed by the C-terminal inner-core/catalytic (IC) domain of human E2p determined at 3.1 Å resolution by cryo electron microscopy (cryoEM). The structure of the N-terminal fragment and four other surface areas of the human E2p IC domain exhibit significant differences from those of the other E2 crystal structures, which may have implications for the integration of E3BP in mammals. This structure also allowed us to obtain a homology model for the highly homologous IC domain of E3BP. Analysis of the interactions of human E2p or E3BP with their adjacent IC domains in the dodecahedron provides new insights into the organization of the E2p/E3BP heterocomplex and suggests a potential contribution by E3BP to catalysis in mammalian PDC.

Co-delivery of human cancer-testis antigens with adjuvant in protein nanoparticles induces higher cell-mediated immune responses.

Nanoparticles have attracted considerable interest as cancer vaccine delivery vehicles for inducing sufficient CD8+ T cell-mediated immune responses to overcome the low immunogenicity of the tumor microenvironment. Our studies described here are the first to examine the effects of clinically-tested human cancer-testis (CT) peptide epitopes within a synthetic nanoparticle. Specifically, we focused on two significant clinical CT targets, the HLA-A2 restricted epitopes of NY-ESO-1 and MAGE-A3, using a viral-mimetic packaging strategy. Our data shows that simultaneous delivery of a NY-ESO-1 epitope (SLLMWITQV) and CpG using the E2 subunit assembly of pyruvate dehydrogenase (E2 nanoparticle), resulted in a 25-fold increase in specific IFN-γ secretion in HLA-A2 transgenic mice. This translated to a 15-fold increase in lytic activity toward target cancer cells expressing the antigen. Immunization with a MAGE-A3 epitope (FLWGPRALV) delivered with CpG in E2 nanoparticles yielded an increase in specific IFN-γ secretion and cell lysis by 6-fold and 9-fold, respectively. Furthermore, combined delivery of NY-ESO-1 and MAGE-A3 antigens in E2 nanoparticles yielded an additive effect that increased lytic activity towards cells bearing NY-ESO-1+ and MAGE-A3+. Our investigations demonstrate that formulation of CT antigens within a nanoparticle can significantly enhance antigen-specific cell-mediated responses, and the combination of the two antigens in a vaccine can preserve the increased individual responses that are observed for each antigen alone.

Reengineering of the human pyruvate dehydrogenase complex: from disintegration to highly active agglomerates.

The pyruvate dehydrogenase complex (PDC) plays a central role in cellular metabolism and regulation. As a metabolite-channeling multi-enzyme complex it acts as a complete nanomachine due to its unique geometry and by coupling a cascade of catalytic reactions using 'swinging arms'. Mammalian and specifically human PDC (hPDC) is assembled from multiple copies of E1 and E3 bound to a large E2/E3BP 60-meric core. A less restrictive and smaller catalytic core, which is still active, is highly desired for both fundamental research on channeling mechanisms and also to create a basis for further modification and engineering of new enzyme cascades. Here, we present the first experimental results of the successful disintegration of the E2/E3BP core while retaining its activity. This was achieved by C-terminal α-helixes double truncations (eight residues from E2 and seven residues from E3BP). Disintegration of the hPDC core via double truncations led to the formation of highly active (approximately 70% of wildtype) apparently unordered clusters or agglomerates and inactive non-agglomerated species (hexamer/trimer). After additional deletion of N-terminal 'swinging arms', the aforementioned C-terminal truncations also caused the formation of agglomerates of minimized E2/E3BP complexes. It is likely that these 'swinging arm' regions are not solely responsible for the formation of the large agglomerates.

Synthesis of diastereomerically pure Lys(Nε-lipoyl) building blocks and their use in Fmoc/tBu solid phase synthesis of lipoyl-containing peptides for diagnosis of primary biliary cirrhosis.

Primary Biliary Cirrhosis is an immune-mediated disease in which one of the epitopes recognized by antimitochondrial autoantibodies is a lipoylated fragment of the PDC-E2 protein. Accordingly, the synthesis of lipoylated peptides as diagnostic tools is a relevant target. Up to now, the proper tools for the introduction of lipoylation on building blocks to be used in Fmoc/tBu solid phase peptide synthesis (SPPS) are lacking, and the role of chirality in lipoylation remains poorly studied. In this paper, we present the synthesis of lipoylated lysine derivatives as pure diastereomeric building blocks suitable for Fmoc/tBu SPPS and their introduction in relevant peptide sequences to possibly serve as synthetic probes for the development of novel diagnostic tools for this disease. The optimization of the synthesis of lipoylated building blocks derived from racemic, (R)-, and (S)-α-lipoic acid is described. Synthesis of peptide probes incorporating lipoylation is described. An insight regarding the cleavage of lipoylated peptides is given, as well as a method to oxidize or reduce the 1,2-dithiolane ring of the lipoyl moiety directly on the peptide without any subsequent purification.

Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.

A novel regulatory mechanism based upon a dynamic core structure for the mitochondrial pyruvate dehydrogenase complex?

The Arabidopsis thaliana genome includes three genes for mitochondrial dihydrolipoamide acetyltransferase, the E2-component of the mitochondrial pyruvate dehydrogenase complex (PDC). Two genes encode E2-proteins with a single lipoyl domain, while the third has a two-lipoyl domain structure. Transcripts for each E2 protein were expressed in all plant organs. Each recombinant AtmtE2 can individually form an icosahedral PDC core structure, and results from bimolecular fluorescence complementation assays are consistent with formation of hetero-core structures from all permutations of the AtmtE2 proteins. We propose a unique regulatory mechanism involving dynamic formation of hetero-core complexes that include both mono- and di-lipoyl forms of AtmtE2.

Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP).

Salt-induced changes in the subunit structure of the Bacillus stearothermophilus lipoate acetyltransferase.

The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C(A)) and a quasi-stable complex (C(Q)). C(A) was larger than C(Q), but smaller than intact E2. C(A) and C(Q), were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2.

Insight to the interaction of the dihydrolipoamide acetyltransferase (E2) core with the peripheral components in the Escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches.

Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 Å resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected reductive acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.

Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation.

Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N-LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N-LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N-LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN-LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N-LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; and (vi) LplA-N-LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.

Applications of small-angle X-ray scattering to biomacromolecular solutions.

Small-angle scattering of X-rays (SAXS) is an established method for low-resolution structural characterization of biological macromolecules in solution. Being complementary to the high resolution methods (X-ray crystallography and NMR), SAXS is often used in combination with them. The technique provides overall three-dimensional structures using ab initio reconstructions and hybrid modeling, and allows one to quantitatively characterize equilibrium mixtures as well as flexible systems. Recent progress in SAXS instrumentation, most notably, high brilliance synchrotron sources, has paved the way for high throughput automated SAXS studies allowing screening of external conditions (pH, temperature, ligand binding etc.). The modern approaches for SAXS data analysis are presented in this review including rapid characterization of macromolecular solutions in terms of low-resolution shapes, validation of high-resolution models in close-to-native conditions, quaternary structure analysis of complexes and quantitative description of the oligomeric composition in mixtures. Practical aspects of SAXS as a standalone tool and its combinations with other structural, biophysical or bioinformatics methods are reviewed. The capabilities of the technique are illustrated by a selection of recent applications for the studies of biological molecules. Future perspectives on SAXS and its potential impact to structural molecular biology are discussed.

The role of natural killer (NK) and NK T cells in the loss of tolerance in murine primary biliary cirrhosis.

One of the major obstacles in dissecting the mechanism of pathology in human primary biliary cirrhosis (PBC) has been the absence of animal models. Our laboratory has focused on a model in which mice, following immunization with a xenobiotic chemical mimic of the immunodominant autoepitope of the E2 component of pyruvate dehydrogenase complex (PDC-E2), develop autoimmune cholangitis. In particular, following immunization with 2-octynoic acid (a synthetic chemical mimic of lipoic acid-lysine located within the inner domain of PDC-E2) coupled to bovine serum albumin (BSA), several strains of mice develop typical anti-mitochondrial autoantibodies and portal inflammation. The role of innate immune effector cells, such as natural killer (NK) cells and that NK T cells, was studied in this model based on the hypothesis that early events during immunization play an important role in the breakdown of tolerance. We report herein that, following in-vivo depletion of NK and NK T cells, there is a marked suppression of anti-mitochondrial autoantibodies and cytokine production from autoreactive T cells. However, there was no change in the clinical pathology of portal inflammation compared to controls. These data support the hypothesis that there are probably multiple steps in the natural history of PBC, including a role of NK and NK T cells in initiating the breakdown of tolerance. However, the data suggest that adaptive autoimmune effector mechanisms are required for the progression of clinical disease.

Complement activation and cell uptake responses toward polymer-functionalized protein nanocapsules.

Self-assembling protein nanocapsules can be engineered for various bionanotechnology applications. Using the dodecahedral scaffold of the E2 subunit from pyruvate dehydrogenase, we introduced non-native surface cysteines for site-directed functionalization. The modified nanoparticle's structural, assembly, and thermostability properties were comparable to the wild-type scaffold (E2-WT), and after conjugation of poly(ethylene glycol) (PEG) to these cysteines, the nanoparticle remained intact and stable up to 79.7 ± 1.8 °C. PEGylation of particles reduced uptake by human monocyte-derived macrophages and MDA-MB-231 breast cancer cells, with decreased uptake as PEG chain length is increased. In vitro C4-depletion and C5a-production assays yielded 97.6 ± 10.8% serum C4 remaining and 40.1 ± 6.0 ng/mL C5a for E2-WT, demonstrating that complement activation is weak for non-PEGylated E2 nanoparticles. Conjugation of PEG to these particles moderately increased complement response to give 79.7 ± 6.0% C4 remaining and 87.6 ± 10.1 ng/mL C5a. Our results demonstrate that PEGylation of the E2 protein nanocapsules can modulate cellular uptake and induce low levels of complement activation, likely via the classical/lectin pathways.

Effects of two solvent conditions on the free energy landscape of the BBL peripheral subunit binding domain.

BBL is a small independently folding domain with two main parallel helices. The experiment of C(α) secondary shifts has shown that changing the pH from ~7 to ~5 results in the reduced helicity at the C-terminus of helix 2. Combining constant pH molecular dynamics with replica exchange, we sampled the protein conformation space and protonation states extensively under a neutral pH condition and an acidic condition. Our results reveal that the solvent conditions influence the free energy landscape. Under the neutral pH condition, the denatured state and the native state are well separated. The condition of the acidic pH reshapes the free energy surface, leading to a broadly populated denatured-state basin and a low free energy barrier between the denatured state and the native state. The acidic pH shifts the equilibrium between the denatured state and the native state in favor of the denatured state. Caution must be used to interpret experimental data under the acidic condition because the contribution of the denatured state is significant. Our simulation results are supported by the fact that the calculated chemical shifts are in good agreement with the experiment data.

The multi-hit hypothesis of primary biliary cirrhosis: polyinosinic-polycytidylic acid (poly I:C) and murine autoimmune cholangitis.

A void in understanding primary biliary cirrhosis (PBC) is the absence of appropriate animal models. Our laboratory has studied a murine model of autoimmune cholangitis induced following immunization with 2-octynoic acid (2OA), an antigen identified following extensive quantitative structural activity relationship (QSAR) analysis, using human autoantibodies and three-dimensional analysis of the mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). Mice immunized with 2OA coupled to bovine serum albumin (BSA) develop anti-mitochondrial antibodies (AMAs) of the identical specificity as humans with PBC, and in addition develop inflammatory portal cell infiltrates in liver. However, the natural history of disease is less severe than in humans and does not include fibrosis. Data from human and autoimmune murine models suggest that environmental and/or infectious agents can exacerbate autoimmune reactions, and a model of PBC has been described in which polyinosinic-polycytidylic acid (poly I:C), a viral RNA mimetic and Toll-like receptor 3 (TLR-3) agonist induces low-titre AMAs and in mild portal infiltrates. We took advantage of our established model to determine whether immunization with 2OA-BSA coupled with poly I:C alters the disease process. Indeed, the addition of poly I:C produces a profound exacerbation of autoimmune cholangitis, including a significant increase in CD8(+) infiltrating T cells, as well as a marked increase of proinflammatory cytokines. In addition, mice have evidence of fibrosis. These findings lend support to the concept that besides breakdown of self-tolerance, there is a requirement of a second 'hit' during the breakdown process that leads to disease which more faithfully mimics human PBC.

Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly.

Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1-E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric 'core' provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an 'average' 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.