Comparative epitope mapping of murine monoclonal and human autoantibodies to human PDH-E2, the major mitochondrial autoantigen of primary biliary cirrhosis.

Immunization with recombinant human pyruvate dehydrogenase (PDH)-E2, the major autoantigen of primary biliary cirrhosis, readily induces a vigorous murine antibody response but does not generate hepatic disease. To determine the fine specificity of this response, 18 mAb were generated from three strains of mice and the reactive epitopes mapped. An initial examination of mAb suggested that they behaved similarly to the antimitochondrial autoantibodies in primary biliary cirrhosis (PBC) because i) all polyclonal antisera and 2 of 18 mAb reacted with all species of mammalian PDH-E2 examined including mouse PDH-E2, ii) 15 of 18 mAb inhibited PDH enzyme function, and iii) the reactivity of mAb toward rPDH-E2 were blocked by PBC sera. However, fine examination of the reactive sequences of the PDH-E2 complex revealed that antibodies identical to those in PBC patients were not produced by experimental immunization. In contrast to PBC, none of the mAb or murine polyclonal sera were able to react with protein X, a lipoic acid-containing component of the PDH complex previously shown to cross-react with PDH-E2 when probed with PBC sera. Although the epitopes for 12 mAb were localized within the inner lipoyl domain, none reacted with mouse PDH-E2 or cross-reacted with the outer lipoyl domain as observed in PBC. In addition, the epitopes of the two mAb which did react with all mammalian species of mitochondria were not localized within the PBC epitope. These findings indicate the highly immunogenic nature of the inner lipoyl domain of PDH-E2. The inability to elicit antibodies of the same specificity in mice, considered together with the highly localized autoantibody response in humans, suggests that antimitochondrial autoantibodies are most likely the result of specific breakdown of tolerance to a unique autoepitope.     

Antimitochondrial antibodies of primary biliary cirrhosis recognize dihydrolipoamide acyltransferase and inhibit enzyme function of the branched chain alpha-ketoacid dehydrogenase complex

Antimitochondrial antibodies (AMA) recognizing the acetyltransferase (E2) of the pyruvate dehydrogenase (PDH) complex have been previously well-documented and the immunodominant epitope mapped. In this study, we demonstrate that sera from patients with primary biliary cirrhosis (PBC) react with another lipoic acid containing acyltransferase enzyme, namely the E2 of the branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Indeed, 85/120 (71%) sera from patients with PBC reacted with BCKD-E2 by immunoblotting against purified BCKD complex. In contrast, sera from patients with chronic active hepatitis or progressive sclerosing cholangitis as well as sera from healthy volunteers did not react with any component enzymes of the BCKD complex. More importantly, BCKD enzyme activity was inhibited after incubation of the BCKD complex with either PBC sera against BCKD-E2 or with affinity purified antisera to BCKD-E2. Enzyme activity was unaltered by control sera or with PBC sera that reacted with PDH-E2 but not BCKD-E2. Furthermore, immunoblots of purified mitochondria probed with PBC sera absorbed with BCKD-E2 demonstrated that BCKD-E2 and PDH-E2 are each recognized by distinct AMA populations which do not cross-react. In addition, affinity purified PBC sera against BCKD-E2 did not react with PDH-E2 nor inhibit PDH enzyme activity, thus providing further evidence that BCKD-E2 and PDH-E2 are recognized by separate AMA. These data further suggest that the BCKD-E2 epitope recognized by AMA contains, or is close to, a functional domain of this enzyme. The availability of cDNA clones encoding BCKD-E2 and PDH-E2 will allow the study of how key metabolic enzymes may be involved in the immunology and pathology of PBC.     

Immunoreactivity of organic mimeotopes of the E2 component of pyruvate dehydrogenase: connecting xenobiotics with primary biliary cirrhosis

In primary biliary cirrhosis (PBC), the major autoepitope recognized by both T and B cells is the inner lipoyl domain of the E2 component of pyruvate dehydrogenase. To address the hypothesis that PBC is induced by xenobiotic exposure, we took advantage of ab initio quantum chemistry and synthesized the inner lipoyl domain of E2 component of pyruvate dehydrogenase, replacing the lipoic acid moiety with synthetic structures designed to mimic a xenobiotically modified lipoyl hapten, and we quantitated the reactivity of these structures with sera from PBC patients. Interestingly, antimitochondrial Abs from all seropositive patients with PBC, but no controls, reacted against 3 of the 18 organic modified autoepitopes significantly better than to the native domain. By structural analysis, the features that correlated with autoantibody binding included synthetic domain peptides with a halide or methyl halide in the meta or para position containing no strong hydrogen bond accepting groups on the phenyl ring of the lysine substituents, and synthetic domain peptides with a relatively low rotation barrier about the linkage bond. Many chemicals including pharmaceuticals and household detergents have the potential to form such halogenated derivatives as metabolites. These data reflect the first time that an organic compound has been shown to serve as a mimeotope for an autoantigen and further provide evidence for a potential mechanism by which environmental organic compounds may cause PBC.     

A lipoyl synthetic octadecapeptide of dihydrolipoamide acetyltransferase specifically recognized by anti-M2 autoantibodies in primary biliary cirrhosis.

Close to 95% of patients with established clinical, biochemical and histologic features of primary biliary cirrhosis (PBC) possess antimitochondrial M2 antibodies reacting with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We examined the ability of synthetic peptides of E2 to be recognized in ELISA by sera from patients with PBC and autoimmune-related disorders. Sera from 14 PBC M2+ patients, 1 PBC M2- patient, 5 non-PBC M2+ patients, and 6 patients with chronic active hepatitis were studied. Among the seven E2 synthetic peptides tested (namely peptides 87-119, 167-184, 169-202, 267-302, 456-477, 498-513 and 530-543), only peptide 167-184 used as OVA conjugate and prepared with lipoic acid (LA) located on lysine 173 (natural inner lipoyl-binding site) was recognized in direct ELISA by PBC M2+ sera. The conjugated peptide 167-184 LA was not recognized in direct ELISA by non-PBC M2+ sera or by sera from patients with chronic active hepatitis. The free peptide 167-184 LA inhibited the ELISA reaction of PBC antibodies to PDH and totally abolished the typical immunofluorescence reaction of PBC sera on rat kidney, stomach and liver, or human HEp-2 cell substrates. No inhibition of ELISA or immunofluorescence reaction was found with the other E2 fragments including peptide 167-184 without LA. Our results show that the lipoyl moiety forms an integral part of a dominant conformational epitope recognized by PBC sera. Inasmuch as the peptide 167-184 LA was not recognized by non-PBC sera in direct ELISA, it could be used as a valuable probe for PBC diagnosis.     

Immunogenetic analysis reveals that epitope shifting occurs during B-cell affinity maturation in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a liver disease characterized by serum autoantibodies against the pyruvate dehydrogenase complex (PDC) located in the inner mitochondrial membrane. The predominant target in PDC has previously been localized to the inner lipoyl domain (ILD) of the E2 subunit. The etiology of PBC is unknown, although molecular mimicry with bacterial PDC has been proposed. Here, we have investigated the etiology of PBC and nature of the autoimmune response by analyzing the structure of a human monoclonal antibody with ILD specificity. Mutants of the monoclonal antibody, which was originally isolated from a patient with PBC, were expressed as Fab by phage display, and tested for reactivity against recombinant domains of the E2 subunit. Fab in which the V(H)-encoded portions were reverted to germline lost reactivity against the ILD alone, but recognized a different epitope in a didomain construct encompassing the ILD, hinge region and E1/E3 binding domain. The complete V(H) and V(L )germline revertant was unreactive with the human ILD and didomain, the Escherichia coli didomain, and whole PDC. We hypothesize that the IgM on the surface of the na?ve B-cell first recognizes an as yet unidentified antigen, and that accumulation of somatic mutations results in an intermolecular epitope shift directed towards an epitope involving the E1/E3 binding domain. Further mutations result in the specificity being redirected to the ILD. These findings also suggest that bacterial molecular mimicry is not involved in initiating disease.     

Prediction of the immunodominant epitope of the pyruvate dehydrogenase complex E2 in primary biliary cirrhosis using phage display

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by autoantibodies reactive with the pyruvate dehydrogenase complex. A conformational epitope has been mapped to aa 91-227 within the inner lipoyl domain of the E2 subunit (pyruvate dehydrogenase complex E2 (PDC-E2)). We have used phage display to further localize this epitope. A random heptapeptide library was screened using IgG from two patients with PBC, with negative selection using pooled normal IgG. Phage that contained peptide inserts (phagotopes) selected using PBC sera differed from those selected using IgG from patients with RA or polychondritis. Two motifs occurred only among the PBC-selected phagotopes; these were MH (13 sequences, 16 phagotopes) and FV (FVEHTRW, FVEIYSP, FVLPWRI). The phagotopes selected were tested for reactivity with anti-PDC-E2 affinity purified from four patients with PBC. Phagotopes that contained 1 of 15 different peptide sequences were reactive with one or more of these four anti-PDC-E2 preparations, whereas phagotopes that contained 1of the remaining 28 sequences were negative. The peptides (FVLPWRI, MHLNTPP, MHLTQSP) encoded by three phagotopes that were strongly reactive with all four preparations of anti-PDC-E2 were synthesized. Each of the selected peptides, but not an irrelevant peptide, inhibited the reactivity by ELISA of PBC serum with recombinant PDC-E2 and reduced the inhibition of the enzyme activity of PDC by a PBC serum. The peptide sequences, along with the known NMR structure of the inner lipoyl domain of PDC-E2, allow the prediction of nonsequential residues 131HM132 and 178FEV180 that contribute to a conformational epitope.     

Functional analysis of the domains of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae

The LAT1 gene encoding the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae was disrupted, and the lat1 null mutant was used to analyze the structure and function of the domains of E2. Disruption of LAT1 did not affect the viability of the cells. Apparently, flux through the PDH complex is not required for growth of S. cerevisiae under the conditions tested. The wild-type and mutant PDH complexes were purified to near-homogeneity and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme assays. Mutant cells transformed with LAT1 on a unit-copy plasmid produced a PDH complex very similar to that of the wild-type PDH complex. Deletion of most of the putative lipoyl domain (residues 8-84) resulted in loss of about 85% of the overall activity, but did not affect the acetyltransferase activity of E2 or the binding of pyruvate dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), and protein X to the truncated E2. Similar results were obtained by deleting the lipoyl domain plus the first hinge region (residues 8-145) and by replacing lysine-47, the putative site of covalent attachment of the lipoyl moiety, by arginine. Although the lipoyl domain of E2 and/or its covalently bound lipoyl moiety were removed, the mutant complexes retained 12-15% of the overall activity of the wild-type PDH complex. Replacement of both lysine-47 in E2 and the equivalent lysine-43 in protein X by arginine resulted in complete loss of overall activity of the mutant PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization with a xenobiotic 6-bromohexanoate bovine serum albumin conjugate induces antimitochondrial antibodies

The E2 subunit of pyruvate dehydrogenase complex (PDC-E2) is the major autoantigen recognized by antimitochondrial Abs (AMA) in primary biliary cirrhosis (PBC). Recently, we replaced the lipoic acid moiety of PDC-E2 with a battery of synthetic structures designed to mimic a xenobiotically modified lipoyl hapten on a 12-aa peptide that was found within the immunodominant autoepitope of PDC-E2 and demonstrated that AMA in PBC reacted against several organic modified mimotopes as well as, or sometimes significantly better than, the native lipoyl domain. Based on this data, we immunized rabbits with one such xenobiotic organic compound, 6-bromohexanoate, coupled to BSA. One hundred percent of immunized rabbits developed AMA that have each and every characteristic of human AMAs with reactivity against PDC-E2, E2 subunit of branched chain 2-oxo-acid dehydrogenase, and E2 subunit of 2-oxoglutarate dehydrogenase complex. The rabbit AMA also inhibited enzymatic function of PDC-E2 and, importantly, binds to peptide sequences not present in the xenobiotic carrier immunogen. In contrast, BSA-immunized controls did not produce such activity. Our observation that animals immunized with a xenobiotic BSA complex produce autoantibodies that react not only with the xenobiotic, but also with mitochondrial autoantigens recognized by autoimmune PBC sera, suggests that environmental xenobiotic agents can be a risk factor for the induction of PBC.     

Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.     

Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T cell response of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease that includes the presence of lymphoid infiltrates in portal tracts, high titer autoantibodies against pyruvate dehydrogenase-E2 (PDH-E2) and branched chain ketoacid dehydrogenase-E2 (BCKD-E2), and biliary tract destruction. The mechanism by which the autoimmune response is induced, the specificity of damage to the biliary epithelium, and the role of T cells in PBC are still unknown. To address these issues, we have taken advantage of a mouse mAb, coined C355.1, and studied its reactivity against a panel of liver tissue from normal subjects as well as a panel of liver specimens from patients with PBC, progressive sclerosing cholangitis, and chronic active hepatitis (CAH). C355.1, much like human autoantibodies to PDH-E2, reacts exclusively by immunoblotting with PDH-E2, binds to the inner lipoyl domain of the protein, and inhibits PDH-E2 activity in vitro. In addition, we have also attempted to develop cloned T cell lines that react with PDH-E2 and/or BCKD-E2 using liver biopsies from patients with PBC, compared with CAH. Although monoclonal C355.1 produced typical mitochondrial fluorescence on sections of normal liver, pancreas, lung, heart, thyroid, and kidney, it produced a distinct and intense reactivity when used to stain the bile ducts of patients with PBC. Nine of 13 PBC liver biopsies studied herein contained bile ducts on light microscopy, all of which reacted intensely at a 1:100 culture supernatant dilution of monoclonal C355.1. In contrast, although bile ducts of liver specimens from normals, CAH, and progressive sclerosing cholangitis also reacted with C355.1, such reactivity was exclusively mitochondrial and readily detectable only at a dilution of 1:2. More importantly, we generated CD4+, CD8-, alpha beta TCR+ cloned T cell lines from patients with PBC, but not from CAH, that produced IL-2 specifically in response to PDH-E2 or BCKD-E2.     

Determination of the critical concentration required for desmin assembly.

The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx. 80 amino acid residues. These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits. A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product. A method has been devised for purifying the three types of independently folded domain from crude extracts of E. coli, based on their pH-(and heat-)stabilities. The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity. This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution.     

Three genes for enzymes of the pyruvate dehydrogenase complex map to human chromosomes 3, 7, and X.

The genes for three proteins of the pyruvate dehydrogenase (PDH) complex have been assigned to human chromosomes by Southern analysis of a panel of human-rodent somatic cell hybrid DNAs with cDNA probes for these genes. PDH-E1 alpha has been localized on human chromosome 3p13-q23. The assignments of lipoamide dehydrogenase(E3) and PDH-E1 alpha [corrected] to chromosomes 7 and Xp, respectively, have been confirmed. Restrictive-fragment-length polymorphisms have been identified with E3, which will permit further localization of this gene by genetic linkage analysis.     

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.     

Disruption and mutagenesis of the Saccharomyces cerevisiae PDX1 gene encoding the protein X component of the pyruvate dehydrogenase complex

Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.     

Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: binding region for the kinase.

A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.     

Structural requirements within the lipoyl domain for the Ca2+-dependent binding and activation of pyruvate dehydrogenase phosphatase isoform 1 or its catalytic subunit

The inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2) 60-mer forms a Ca(2+)-dependent complex with the pyruvate dehydrogenase phosphatase 1 (PDP1) or its catalytic subunit, PDP1c, in facilitating large enhancements of the activities of PDP1 (10-fold) or PDP1c (6-fold). L2 binding to PDP1 or PDP1c requires the lipoyl-lysine prosthetic group and specificity residues that distinguish L2 from the other lipoyl domains (L1 in E2 and L3 in the E3-binding component). The L2-surface structure contributing to binding was mapped by comparing the capacities of well folded mutant or lipoyl analog-substituted L2 domains to interfere with E2 activation by competitively binding to PDP1 or PDP1c. Our results reveal the critical importance of a regional set of residues near the lipoyl group and of the octanoyl but not the dithiolane ring structure of the lipoyl group. At the other end of the lipoyl domain, substitution of Glu(182) by alanine or glutamine removed L2 binding to PDP1 or PDP1c, and these substitutions for the neighboring Glu(179) also greatly hindered complex formation (E179A > E179Q). Among 11 substitutions in L2 at sites of major surface residue differences between the L1 and L2 domains, only the conversion of Val-Gln(181) located between the critical Glu(179) and Glu(182) to the aligned Ser-Leu sequence of the L1 domain greatly reduced L2 binding. Certain modified L2 altered E2 activation of PDP1 differently than PDP1c, supporting significant impact of the regulatory PDP1r subunit on PDP1 binding to L2. Our results indicate hydrophobic binding via the extended aliphatic structure of the lipoyl group and required adjacent L2 structure anchor PDP1 by acting in concert with an acidic cluster at the other end of the domain.     

Site-directed mutagenesis of a loop at the active site of E1 (alpha2beta2) of the pyruvate dehydrogenase complex. A possible common sequence motif.

Limited proteolysis of the pyruvate decarboxylase (E1, alpha2beta2) component of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1alpha subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the alpha-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1alpha chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an "open-close" mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).     

Configuration of interdomain linkers in pyruvate dehydrogenase complex of Escherichia coli as determined by cryoelectron microscopy.

The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains). In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC. From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions. To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin. In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core. Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro. Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core. The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3. These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations.     

Domain structures of the dihydrolipoyl transacetylase and the protein X components of mammalian pyruvate dehydrogenase complex. Selective cleavage by protease Arg C

Treatment of the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2-X-KcKb) with protease Arg C selectively converted protein X into an inner domain fragment (Mr approximately equal to 35,000) and an outer (lipoyl-bearing) domain fragment (Mr approximately equal to 15,500). These fragments were larger and much smaller, respectively, than the inner domain and outer domain fragments derived from the E2 component, supporting the conclusion that protein X is distinct from the E2 component. Protease Arg C cleaved the Kb subunit more slowly than protein X. An increase in kinase activity correlated with this cleavage of the Kb subunits. An even slower cleavage of E2 subunits generated an inner domain fragment (Mr approximately equal to 31,500) and a lipoyl-bearing domain fragment (Mr approximately equal to 49,000) which had Mr values at least 3,000 and 10,000 larger, respectively, than the corresponding E2 fragments generated by trypsin treatment of the subcomplex. Following various extents of cleavage with protease Arg C or trypsin, residual oligomeric subcomplexes were isolated and characterized. We found that selective removal of the lipoyl-bearing domain of protein X did not alter lipoyl-mediated regulation of the kinase indicating that the lipoyl residues bound to E2 subunits are effective, that the inner domain of protein X remained associated with the inner domain of E2 subunits following the complete removal of the outer domains of both E2 and protein X, that, with only 10% of the E2 subunits intact, nearly half of the catalytic (Kc) subunits of the kinase were bound by the residual subcomplex, and that removal of the remaining outer domains from E2 subunits released the Kc subunits. Thus, protein X is unique among the subunits of the complex in binding tightly to the oligomeric inner domain of the transacetylase, and the outer domain of the transacetylase serves to bind to and facilitate the regulation of the catalytic subunit of the kinase.