Segmental structure and protein domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Genetic reconstruction in vitro and 1H-n.m.r. spectroscopy.

A deletion in vitro can be made in the aceEF-lpd operon encoding the pyruvate dehydrogenase multienzyme complex of Escherichia coli, which causes deletion of two of the three homologous lipoyl domains that comprise the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain. An active complex is still formed and 1H-n.m.r. spectroscopy of this modified complex revealed that many of the unusually sharp resonances previously attributed to conformationally mobile segments in the wild-type E2p polypeptide chains had correspondingly disappeared. A further deletion was engineered in the long (alanine + proline)-rich segment of polypeptide chain that linked the one remaining lipoyl domain to the C-terminal half of the E2p chain. 1H-n.m.r. spectroscopy of the resulting enzyme complex, which was also active, revealed a further corresponding loss in the unusually sharp resonances observed in the spectrum. These experiments strongly support the view that the sharp resonances derive, principally at least, from the three long (alanine + proline)-rich sequences which separate the three lipoyl domains and link them to the C-terminal half of the E2p chain. Closer examination of the 400 MHz 1H-n.m.r. spectra of the wild-type and restructured complexes, and of the products of limited proteolysis, revealed another sharp but smaller resonance. This was tentatively attributed to another, but smaller, (alanine + proline)-rich sequence that separates the dihydrolipoamide dehydrogenase-binding domain from the inner core domain in the C-terminal half of the E2p chain. If this sequence is also conformationally flexible, it may explain previous fluorescence data which suggest that dihydrolipoamide dehydrogenase bound to the enzyme complex is quite mobile. The acetyltransferase active site in the E2p chain was shown to reside in the inner core domain, between residues 370 and 629.     

Limited proteolysis and proton NMR spectroscopy of Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.     

Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the ¹H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin–spin relaxation times, T₂, with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the ¹H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by ¹H n.m.r. spectroscopy.     

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

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

Sequence-specific 1H-NMR assignments and secondary structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex

The lipoyl domain (residues 1-85) of the lipoate-acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been subjected to detailed structural analysis by means of two-dimensional (2D) 1H-NMR spectroscopy at 400 MHz. Sequence-specific proton resonance assignments were made, but at this field strength not all of the side-chain protons could be assigned, especially from complex spin systems like those of leucine, proline and lysine residues. Measurement of short-range interproton distances identified two extensive regions of beta-sheet, each containing four anti-parallel peptide strands. The lipoyl-lysine residue (Lys42) is located in a tight turn at a corner of one sheet, the N-terminal and C-terminal residues of the domain are close together in two adjacent beta-strands in the other. The lipoylated and unlipoylated forms of the domain have almost identical spectra, indicating that there is little, if any, conformational change in the protein as a result of the post-translational modification.     

Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides.

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.     

Temperature-dependent structure of the E x S complex of Bacillus stearothermophilus alcohol dehydrogenase

The catalytic chemistry of the thermophilic Bacillus stearothermophilus alcohol dehydrogenase (HtADH) closely resembles that of mesophilic horse liver alcohol dehydrogenase (HLADH). Molecular dynamics (MD) simulations of the htADH x NAD+ x EtO- complex at 298, 323, and 348 K show that the structure of the ligated Zn2+...EtO- complex varies slightly with change in temperature. The MD-created Boltzmann distribution of htADH x NAD+ x EtO- structures establishes the formation of multiple states which increase in number with a decrease in temperature. The motions of the cofactor domain are highly correlated with the motions of NAD+ at the optimal growth temperature (348 K), with NAD+ being pushed toward the substrate by Val260. With a decrease in temperature, the motion together of the cofactor and substrate is reversed, and at 298 K, the nicotinamide ring of the cofactor moves away from the substrate. Both the distance between and the angle of approach of C4 of NAD+ and HD of EtO- become distorted from those of the reactive conformation. The percentages of ground state present as the reactive conformation at different temperatures are approximately correlated with the kcat for the htADH enzymatic reaction. The rate constant for the htADH x NAD+ x EtOH --> htADH x NAD+ x EtO- proton dissociation, which is mediated by Thr40-OH, becomes slower at lower temperatures. The time-dependent distance between EtO- and Thr40-OH reveals that the Thr40 hydroxyl group sways between the substrate and NAD+ ribose 2'-hydroxyl group at the optimal enzyme growth temperature, and this movement is effectively frozen out as the temperature decreases. The temperature dependence of active site conformations is due to the change in both long-range and short-range motions of the E x S complex.     

Thermally induced changes of lipoate acetyltransferase inner core isolated from the Bacillus stearothermophilus pyruvate dehydrogenase complex

Incubation at 70 degrees C converted the Bacillus stearothermophilus lipoate acetyltransferase inner core into an unidentified active molecular form, X, yielding an inactive aggregate. The core and X showed similar thermostabilities, but they were different in the recovery of enzyme activity after incubation with 1.2-2.0 M guanidine hydrochloride and its subsequent removal; the core was hardly recovered, but X was well recovered.     

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.     

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.     

1H-n.m.r. studies of squash seed trypsin inhibitor

1H-n.m.r. studies at 500 MHz have been performed on a trypsin inhibitor (CMTI-III) found in squash seed (Cucurbita maxima). The sequential resonance assignments have been made using two-dimensional techniques. The chemical shifts for the assigned protons are reported at 30 degrees, pH 2.8 and form a basis for the determination of the solution structure of CMTI-III. Analysis of the NOE data, NH-alpha CH vicinal coupling constants and pattern of slowly exchanging amide protons indicates that the predominant feature of the solution conformation is a triple stranded beta sheet consisting of residues 8-10, 21-23, and 26-29. Residues 12-15 appear to form a beta turn.     

Expression in Escherichia coli of a sub-gene encoding the lipoyl domain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus

A sub-gene encoding the lipoyl domain (residues 1-85) of the lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. Approx. 80% of the domain was unlipoylated but most of the remainder was correctly lipoylated on Lys-42 and could be reductively acetylated by the B stearothermophilus enzyme complex. A small proportion (approx. 4%) of the domain carried an aberrant substituent, possibly an octanoyl group, on Lys-42. The 400 MHz 1H NMR spectra of the lipoylated and unlipoylated domains were essentially identical and closely resembled that of the native lipoyl domain.