Direct evidence for the size and conformational variability of the pyruvate dehydrogenase complex revealed by three-dimensional electron microscopy. The "breathing" core and its functional relationship to protein dynamics

Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE(2)) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE(2) forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE(2) in the same field vary in size approximately 20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-A difference in the diameter of the smallest and largest structures that corresponds to approximately 14 A of variation in the length of the bridge between interconnected trimers. Companion studies of mature E(2) show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE(2) shows that there is an approximately 10-A gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme 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.     

Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase

The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution. The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms. The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active-site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o.     

Nucleotide sequence of a cDNA for the dihydrolipoamide acetyltransferase component of human pyruvate dehydrogenase complex

Deoxynucleotide sequencing of a cDNA for the dihydrolipoamide acetyltransferase (PDC-E₂) component of human pyruvate dehydrogenase complex (PDC) revealed an open reading frame of 1848 base pairs corresponding to a leader sequence of 54 amino acids and a mature protein of 561 amino acids (59 551 Da). Both an amino-terminal lipoyl-bearing domain and a carboxy-terminal catalytic domain are present in the deduced amino acid sequence. The lipoyl-bearing domain contains two repeating units of 127 amino acids, each harboring one lipoic acid-binding lysine. Thus, mammalian PDC-E₂ differs as to the number of lipoic acid-binding sites from other dihydrolipoamide acyltransferases in both prokaryotic and eukaryotic organisms.     

Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I' domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2.E3BP was thought to be 60 E2 plus approximately 12 E3BP. We have prepared homogenous human components. E2 and E2.E3BP have s(20,w) values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2.E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2.E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound approximately 60 E1 and maximally sedimented 64.4 +/- 1.5 S faster than E2, whereas E1-saturated E2.E3BP maximally sedimented 49.5 +/- 1.4 S faster than E2.E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (approximately 12) were bound by E2.E3BP than by E2. The findings of a smaller E2.E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I' domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248.E3BP12 mass, which is consistent with this model.     

Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine

Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 A structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.     

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.     

Solution Structure and Characterisation of the Human Pyruvate Dehydrogenase Complex Core Assembly

Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2 + E3BP) core organisation: the ‘addition’ model (60 + 12) and the ‘substitution’ model (48 + 12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the ‘substitution’ model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.     

Extended polypeptide linkers establish the spatial architecture of a pyruvate dehydrogenase multienzyme complex

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.     

The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain. By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain. The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration. The lipoyl domain is monomeric with a mass of 32.6 kDa. It is an elongated structure with f/fo = 1.62. Circulair dichroic studies indicates little secondary structure. The catalytic domain is polymeric with S20.w = 17 S and mass = 530 kDa. It is a compact structure with f/fo = 1.24 and shows 40% of the secondary structure of E2. The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy. Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa. An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichia coli E2. The catalytic domain possesses the catalytic site, but in contrast to the E. coli subunit binding domain the pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis. From comparison with the E. coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the E2/E2 interaction sites, and the E1 binding site, are indicated.     

Cryoelectron microscopy of mammalian pyruvate dehydrogenase complex.

Cryoelectron microscopy has been performed on frozen-hydrated pyruvate dehydrogenase complexes from bovine heart and kidney and on various subcomplexes consisting of the dihydrolipoyl transacetylase-based (E2) core and substoichiometric levels of the other two major components, pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3). The diameter of frozen-hydrated pyruvate dehydrogenase complex (PDC) is 50 nm, which is significantly larger than previously reported values. On the basis of micrographs of the subcomplexes, it is concluded that the E1 and E3 are attached to the E2-core complex by extended (4-6 nm maximally) flexible tethers. PDC constructed in this manner would probably collapse and appear smaller than its native size when dehydrated, as was the case in previous electron microscopy studies. The tether linking E1 to the core involves the hinge sequence located between the E1-binding and catalytic domains in the primary sequence of E2, whereas the tether linking E3 is probably derived from a similar hinge-type sequence in component X. Tilting of the E2-based cores and comparison with model structures confirmed that their overall shape is that of a pentagonal dodecahedron. The approximately 6 copies of protein X present in PDC do not appear to be clustered in one or two regions of the complex and are not likely to be symmetrically distributed.     

The human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.     

Study of the pyruvate dehydrogenase complex by circular dichroism]

A comparative study of the pyruvate dehydrogenase complex and its pyruvate dehydrogenase component was carried out by using the circular dichroism method. It was found that the spectral properties of the pyruvate dehydrogenase complex are determined by those of its first component: i) the spectrum of the thiamine pyrophosphate-free pyruvate dehydrogenase complex displayed the main characteristics of the pyruvate dehydrogenase component; ii) the appearance of the charge transfer complex band during thiamine pyrophosphate saturation was revealed for the both proteins; iii) in both cases the charge transfer complex band disappeared after the interaction of the holoform with pyruvate and reappeared after the addition of dithiothreitol used as a deacetylating reagent. Coenzyme A in the same reaction selectively deacetylated the pyruvate dehydrogenase complex (but not its pyruvate dehydrogenase component). The spectral dynamics of pyruvate dehydrogenase reflects the functional changes in the enzyme active centers during the catalytic act. The similarity of the spectral behaviour of pyruvate dehydrogenase within the complex structure and in the isolated state provides support for the earlier proposed mechanism of the pyruvate dehydrogenase action and ensures a methodological basis for its direct investigation within the complex structure.     

Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes.

The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.     

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.     

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

The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772–19780).The human pyruvate dehydrogenase complex (PDC)³ resides in mitochondria and catalyzes the oxidative decarboxylation of pyruvate to yield acetyl-CoA and reducing equivalents (NADH), serving as a link between glycolysis and the Krebs cycle (1–3). The PDC is a large (∼9.5 MDa) catalytic machine comprising multiple protein components. The three catalytic components are pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), with E3 being a common component between different α-keto acid dehydrogenase complexes. The two regulatory enzymes in the PDC are the isoforms of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.The PDC is organized around a structural core, which includes the C-terminal domains of E2p and a noncatalytic component that specifically binds E3, i.e. the E3-binding protein (E3BP). To this E2p/E3BP core, multiple copies of the other PDC components are tethered through noncovalent interactions. Each E2p subunit contains two consecutive N-terminal lipoic acid-bearing domains (LBDs), termed L1 and L2, followed by the E1p-binding domain (E1pBD) and the C-terminal inner-core/catalytic domain, with these independent domains connected by unstructured linkers. Similarly, each E3BP subunit consists of a single N-terminal LBD (referred to as L3), the E3-binding domain (E3BD), and the noncatalytic inner core domain. Together, the inner core domains of E2p and E3BP assemble to form the dodecahedral 60-meric E2p/E3BP core. The role of the E1pBD and E3BD domains is to tether E1p and E3, respectively, to the periphery of the E2p/E3BP core. It is presumed that the LBDs (L1, L2, and L3) shuttle between the active sites of the three catalytic components of the PDC during the oxidative decarboxylation cycle (4). The eukaryotic PDC is unique among α-keto acid dehydrogenase complexes in its requirement for E3BP; prokaryotic PDCs employ the single subunit-binding domain to secure either E1p or E3 to the complex (5).Using a “divide-and-conquer” approach, a wealth of structural information on the PDC has been accumulated recently. High-resolution crystal structures are available for the human E1p (6–8) and E3 components (9). A model for the human E2p has been constructed based on an 8.8-Å electron density map available from cryo-electron microscopy (10). Additionally, solution and crystal structures of the L1 and L2 domains of E2p have been determined (11–13), and the high-resolution crystal structures of the E3BD (14, 15), pyruvate dehydrogenase kinase isoforms 1–4 (12, 16–18), and pyruvate dehydrogenase phosphatase isoform 1 (19) are known. Therefore, atomic models are available for almost all components and domains of the mammalian PDC.With the successes of the above structural approach, attention has turned to the overall structure of the PDC. There are two outstanding questions as follows. What are the subunit and overall catalytic component stoichiometries? What are the positions and orientations of the components in this large catalytic machine? Yu et al. (10) recently determined the cryo-EM structure of a PDC core comprising only human E2p subunits. Like yeast E2p, human E2p adopts a dodecahedral structure composed of 60 E2p proteins; each face of the dodecahedron has a large gap. Although this structure is highly informative, the composition of this core deviates substantially from that of the native PDC, because no E3BP subunits are present in the core structure. Based on the similar structure of the dodecahedral yeast PDC, a hypothesis was formed that, in human PDC, 12 copies of E3BP bind in the 12 gaps, which is termed the “60/12” model (20). Biophysical studies on complexes of E2p and E3BP later negated the 60/12 model; Hiromasa et al. (21) therefore posited an alternative, the “48/12” model, in which the dodecahedral core includes 48 E2p subunits and 12 E3BP proteins. A further source of conjecture is how many E1p and E3 components bind to the periphery of the PDC. If one binding domain binds to one peripheral catalytic component, a maximally occupied 60/12 PDC would harbor 60 E1p heterotetramers and 12 E3 dimers (or 48 E1ps and 12 E3s in the 48/12 model). The notion of such 1:1 binding is supported by the preponderance of available biophysical evidence. Specifically, two crystal structures, site-directed mutagenesis, and calorimetric measurements describe a 1:1 interaction between E3BD and E3 (14, 15). Also, although no structures are available for the human E1p-E1pBD complex, a crystal structure of the homologs of these proteins from Bacillus stearothermophilus also demonstrates a 1:1 interaction between the E1pBD of E2p and the E1p heterotetramer (22). In addition, ITC experiments performed on the bacterial E1p and the cognate subunit-binding domain indicate a 1:1 association (23). At variance with the above observations, a different subunit stoichiometry has been proposed by Smolle et al. (24, 25). Their evidence suggests that two binding domains bind for every peripheral component; such an arrangement potentially yields a PDC with half as many peripheral components bound.This study was undertaken to ascertain the subunit and component stoichiometries of the human PDC, particularly with regard to interactions between the E3BD and the E3 dimer. We show that quantification of bands on an SDS-polyacrylamide gel of a PDC reconstituted at saturating E1p and E3 concentrations supports neither the 60/12 nor the 48/12 model. Instead, a “40/20” model is proposed, and subsequent ITC and analytical ultracentrifugation (AUC) data corroborate this new model. In addition, results from electrophoretic mobility shift assays, ITC, and AUC presented here uniformly show a 1:1 interaction between E3BD and the E3 dimer as well as between E1pBD and the E1p heterotetramer. The implications of this 1:1 binding stoichiometry for the macromolecular assembly of the PDC are discussed.     

Chain folding in the dihydrolipoyl acyltransferase components of the 2-oxo-acid dehydrogenase complexes from Escherichia coli. Identification of a segment involved in binding the E3 subunit

The state of assembly of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes was examined after the dihydrolipoyl acyltransferase (E2) component of each enzyme system had been subjected to varying degrees of limited proteolysis. Dissociation of the dihydrolipoyl dehydrogenase (E3) component accompanied specifically the excision of a homologous segment of each E2 chain that connects the N-terminal lipoyl domain(s) with a C-terminal catalytic domain. The latter remains aggregated as a 24-mer and retains its capacity to bind the 2-oxo-acid decarboxylase (E1) component. The relevant segment of the E2o chain from the 2-oxoglutarate dehydrogenase complex was isolated and shown to be a folded protein which still binds to E3.     

Site-directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Binding of the peripheral components E1p and E3.

Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex. Upon binding of peripheral components, the 24-subunit core of A. vinelandii wild-type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers. Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (delta P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli. Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes. It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.     

Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.     

Dihydrolipoamide dehydrogenase: functional similarities and divergent evolution of the pyridine nucleotide-disulfide oxidoreductases

Dihydrolipoamide dehydrogenase (E3) is the common component of the three alpha-ketoacid dehydrogenase complexes oxidizing pyruvate, alpha-ketoglutarate, and the branched-chain alpha-ketoacids. E3 also participates in the glycine cleavage system. E3 belongs to the enzyme family called pyridine nucleotide-disulfide oxidoreductases, catalyzing the electron transfer between pyridine nucleotides and disulfide compounds. This review summarizes the information available for E3 from a variety of species, from a halophilic archaebacterium which has E3 but no alpha-ketoacid dehydrogenase complexes, to mammalian species. Evidence is reviewed for the existence of two E3 isozymes (one for pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex and the other for branched-chain alpha-ketoacid dehydrogenase complex) in Pseudomonas species and for possible mammalian isozymes of E3, one associated with the three alpha-ketoacid dehydrogenase complexes and one for the glycine cleavage system. The comparison of the complete amino acid sequences of E3 from Escherichia coli, yeast, pig, and human shows considerable homologies of certain amino acid residues or short stretches of sequences, especially in the specific catalytic and structural domains. Similar homology is found with the limited available amino acid sequence information on E3 from several other species. Sequence comparison is also presented for other member flavoproteins [e.g., glutathione reductase and mercury(II) reductase] of the pyridine nucleotide-disulfide oxidoreductase family. Based on the known tertiary structure of human glutathione reductase it may be possible to predict the domain structures of E3. Additionally, the sequence information may help to better understand a divergent evolutionary relationship among these flavoproteins in different species.