Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl). The pattern of changes in the values of DeltaG degrees, DeltaH degrees and TDeltaS degrees that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropy-enthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.     

The molecular origins of specificity in the assembly of a multienzyme complex

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.     

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.     

Prediction of the binding site on E1 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The beta-subunit (E1beta) of the pyruvate decarboxylase (E1, alpha(2)beta(2)) component of the Bacillus stearothermophilus pyruvate dehydrogenase complex was comparatively modelled based on the crystal structures of the homologous 2-oxoisovalerate decarboxylase of Pseudomonas putida and Homo sapiens. Based on this homology modelling, alanine-scanning mutagenesis studies revealed that the negatively charged side chain of Glu285 and the hydrophobic side chain of Phe324 are of particular importance in the interaction with the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the complex. These results help to identify the site of interaction on the E1beta subunit and are consistent with thermodynamic evidence of a mixture of electrostatic and hydrophobic interactions being involved.     

Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis.

The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (alpha2beta2) of E1alpha and E1beta polypeptide chains. The domain structure of the E1alpha and E1beta chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha2beta2). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.     

Two lipoyl domains in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis

A fragment of DNA incorporating the gene, pdhC, that encodes the dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis was cloned and a DNA sequence of 2360 bp was determined. The pdhC gene (1620 bp) corresponds to an E2 chain of 539 amino acid residues, Mr 56,466, comprising two lipoyl domains, a peripheral subunit-binding domain and an acetyltransferase domain, linked together by regions of polypeptide chain rich in alanine, proline and charged amino acids. The S. faecalis E2 chain differs in the number of its lipoyl domains from the E2 chains of all bacterial pyruvate dehydrogenase complexes hitherto described.     

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

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

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.     

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.     

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

The pyruvate dehydrogenase complex (PDC) from muscle of the adult parasitic nematode Ascaris suum plays a unique role in its anaerobic mitochondrial metabolism. Resolution of the intact complex in high salt dissociates the pyruvate dehydrogenase subunit but leaves the dihydrolipoyl dehydrogenase subunit (E3) and two other proteins with apparent M(r)s of 45 and 43 kDa bound to the dihydrolipoyl transacetylase (E2) core. These proteins are not observable on Coomassie brilliant blue-stained gels of other eukaryotic PDCs, but the 45-kDa protein is similar in apparent M(r), pI, and sensitivity to trypsin to the Kb subunit of the bovine kidney PDH alpha kinase. Acetylation of the ascarid PDC with [2-14C]pyruvate under conditions designed to maximize the incorporation of label into protein yielded only a single radiolabeled subunit, E2. These results confirm earlier reports that the ascarid PDC lacks protein X, an integral component recently identified in other eukaryotic PDCs. About 1.6 to 1.8 mol of 14C was incorporated/mole of E2, suggesting that the ascarid E2 contained two lipoly-bearing domains. Domain mapping of the 14C-acetylated ascarid E2 by limited tryptic digestion identified two lipoyl-bearing fragments with apparent M(r)s of 50 and 34 kDa and two core fragments with apparent M(r)s of 46 and 30 kDa. The ascarid E2 domain structure appears to be similar to that of other E2s. However, it appears that the subunit-binding domain (E2B) of the ascarid E2 may be significantly larger or be flanked by larger than normal interdomain regions. An enlarged E2B domain may be necessary to accommodate the additional binding of E3 to the E2 subunit in the ascarid complex, in the absence of protein X.     

The pyruvate dehydrogenase multienzyme complex

During the review period, several structures of component enzymes and domains of enzymes of this multienzyme complex were determined. Three structures of the flavoprotein component, dihydrolipoamide dehydrogenase, became available. The structure of the core component, dihydrolipoyl acetyltransferase, can in principle be constructed from the known structures of its modules: the lipoyl, the peripheral subunit-binding and the catalytic domain. Dynamic aspects, such as the structure and function of the inter-domain linkers in dihydrolipoyl acetyltransferase and the conformational changes invlved in the mechanism of electron transfer in dihydrolipoamide dehydrogenase, remain to be clarified. Although several questions concerning the structure of the individual components of the complex have been solved, there is still much to learn about the assembly pathway. In mammalian complexes, the structure and function of protein X remains something of a riddle.     

Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis

Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.     

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.     

Molecular structure of a 9-MDa icosahedral pyruvate dehydrogenase subcomplex containing the E2 and E3 enzymes using cryoelectron microscopy

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.     

Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution

The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes.     

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.     

Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Use of a truncated protein domain in NMR spectroscopy

A (15)N-labelled peripheral-subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2p) and the dimer of a solubilized interface domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were used to investigate the basis of the interaction of E2p with E3 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the (15)N and (1)H amide chemical shifts when the PSBD forms a 1 : 1 complex with E3int. All of the 13 amino acids reside near the N-terminus of helix I of PSBD or in the loop region between helix II and helix III. (15)N backbone dynamics experiments on PSBD indicate that the structured region extends from Val129 to Ala168, with limited structure present in residues Asn126 to Arg128. The presence of structure in the region before helix I was confirmed by a refinement of the NMR structure of uncomplexed PSBD. Comparison of the crystal structure of the PSBD bound to E3 with the solution structure of uncomplexed PSBD described here indicates that the PSBD undergoes almost no conformational change upon binding to E3. These studies exemplify and validate the novel use of a solubilized, truncated protein domain in overcoming the limitations of high molecular mass on NMR spectroscopy.     

Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

T(2) relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase component, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first committed and irreversible catalytic step. The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain. The interactions between the free lipoyl domain (9 kDa) and free E1alpha (41 kDa), E1beta (35 kDa) and intact E1alpha(2)beta(2) (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uniformly (15)N-labeled lipoyl domain and unlabeled E1 components. Major contact points on the lipoyl domain were identified from changes in the backbone (15)N spin-spin relaxation time in the presence and absence of E1(alpha(2)beta(2)) or its individual E1alpha or E1beta components. Although the E1alpha subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1alpha and E1beta. A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1. None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain. This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine beta-turn region, as indicated by chemical shift data. Interaction with E1 and subsequent reductive acetylation are not necessarily coupled.     

The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. A reconsideration

After limited proteolysis of the dihydrolipoyl transacetylase component (E2) of Azotobacter vinelandii pyruvate dehydrogenase complex (PDC), a C-terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl-containing N-terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C-terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C-terminus of E2. Together with the previously determined N-terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24-meric structure is concluded instead of the 32-meric structure proposed previously. From the effect of guanidine hydrochloride on the light-scattering of intact E2 it was concluded that dissociation occurs in a two-step reaction resulting in particles with an average mass 1/6 that of the original mass before the N----D transition takes place. Cross-linking experiments with the catalytic domain indicated that the multimeric E2 is built from tetramers and that the tetramers are arranged as a dimer of dimers. A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A. vinelandii.     

Substrate-induced structural changes of the pyruvate dehydrogenase multienzyme complex

The time course of the overall reaction catalyzed by the pyruvate dehydrogenase multienzyme complex produces an unexpectedly high lag (tau = 8 S) even in the presence of saturating concentrations of its substrates. The preincubation of the pyruvate dehydrogenase complex with one of the substrates alone decreases the duration of this lag, and all the substrates of the pyruvate dehydrogenase component (E1) and dihydrolipoyl transacetylase component (E2) together (pyruvate, thiamine pyrophosphate, and CoA) result in the complete disappearance of the lag. The reduction of the dihydrolipoyl dehydrogenase component (E3) of the pyruvate dehydrogenase complex with the substrates of the complex in the absence of NAD+ produces significantly different quenching in the FAD fluorescence, and then the reduction with the substrates of E3 as dihydrolipoic acid and dithioerythritol. (The formation of FADH2 was not observed in the system.) The higher fluorescence quenching in the presence of substrates of pyruvate dehydrogenase complex compared to the effect caused by the substrates of the E3 component (dihydrolipoic acid and DTE) indicates conformational changes additionally manifested in the fluorescence properties of the enzyme complex. The substrate-induced quenching of the enzyme-bound FAD fluorescence shows biphasic kinetics. The rate constant of the slow phase is comparable with the rate constant calculated from the time duration of the lag phase observed in the overall reaction. The kinetic analysis of both intensity and anisotropy decrease of the FAD fluorescence suggests a consecutive transmittance of an all substrate-coordinated, induced conformational changes directed from the pyruvate dehydrogenase-via the lipoyl transacetylase--to the lipoyl dehydrogenase. Two simultaneous conformational effects caused by binding of the substrates can be distinguished; one of them results the fluorescence of the bound FAD to be more quenched, while the other makes the FAD more mobile. The first-order rate constants of both these conformational changes were determined. The present observations suggest that the pyruvate dehydrogenase complex exists in a partially inactive state in the absence of its substrates, and it becomes active due to conformational changes caused by the binding of its substrates.