Interaction of avidin with the lipoyl domains in the pyruvate dehydrogenase multienzyme complex: three-dimensional location and similarity to biotinyl domains in carboxylases.

Avidin can form intermolecular cross-links between particles of the pyruvate dehydrogenase multienzyme complex from various sources. Avidin does this by binding to lipoic acid-containing regions of the dihydrolipoamide acetyltransferase polypeptide chains that comprise the structural core of the complex. It is inferred that the lipoyl domains of the acetyltransferase chain extend outwards from the interior of the enzyme particle, interdigitating between the subunits of the other two enzymes bound peripherally in the assembled structure, with the lipoyl-lysine residues capable of reaching to within at least 1-2 nm of the outer surface of the enzyme complex (diameter ca. 37 nm). The distribution of enzymic activities between different domains of the dihydrolipoamide acetyltransferase chain implies that considerable movement of the lipoyl domains is a feature of the catalytic activity of the enzyme complex. There is evidence that the lipoyl domain of the 2-oxo acid dehydrogenase complexes is similar in structure to a domain that binds the cofactor biotin, also in amide linkage with a specific lysine residue, in the biotin-dependent class of carboxylases.     

Construction and properties of pyruvate dehydrogenase complexes with up to nine lipoyl domains per lipoate acetyltransferase chain

The lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli have three tandemly repeated lipoyl domains, although net deletions of one or two has no apparent effect on the activity of the purified complexes. Plasmids containing IPTG-inducible aceEF-lpd operons, which encode PDH complexes bearing from one to nine lipoyl domains per E2p chain (24-216 per complex), were constructed. They were all capable of restoring the nutritional lesion of a strain lacking PDH complex and they all expressed active sedimentable multienzyme complexes having a relatively normal range of subunit stoichiometries. The extra domains are presumed to protrude from the E2p core (24-mer) without significantly affecting the assembly of the E1p and E3 subunits on the respective edges and faces of the cubic core. However, the catalytic activities of the overproduced complexes containing four to nine lipoyl domains per E2p chain were lower than those with fewer lipoyl domains. This could be due to under-lipoylation of the domains participating in catalysis and interference from unlipoylated domains.     

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.     

Determination of the critical concentration required for desmin assembly.

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

Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.     

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.     

Structural determinants of post-translational modification and catalytic specificity for the lipoyl domains of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The lipoyl domains of the dihydrolipoyl acyltransferase (E2p, E2o) components of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes are specifically recognised by their cognate 2-oxo acid decarboxylase (E1p, E1o). A prominent surface loop links the first and second beta-strands in all lipoyl domains, close in space to the lipoyl-lysine beta-turn. This loop was subjected to various modifications by directed mutagenesis of a sub-gene encoding a lipoyl domain of Escherichia coli E2p. Deletion of the loop (four residues) rendered the domain incapable of reductive acetylation by E. coli E1p in the presence of pyruvate, but insertion of a new loop (six residues) corresponding to that in the E2o lipoyl domain partly restored this ability, albeit with a much lower rate. However, the modified domain remained unable to undergo reductive succinylation by E1o in the presence of 2-oxoglutarate. Additional exchange of the two residues on the C-terminal side of the loop (V14A, E15T) had no effect. Insertion of a different four-residue loop also restored a limited ability to undergo reductive acetylation, but still significantly less than that of the wild-type domain. Exchanging the residue on the N-terminal side of the lipoyl-lysine beta-turn in the E2p and E2o domains (G39T), both singly and in conjunction with the loop exchange, had no effect on the ability of the E2p domain to be reductively acetylated but did confer a slight increase in susceptibility to reductive succinylation. All mutant E2p domains, apart from that with the loop deletion (LD), were readily lipoylated in vitro by E. coli lipoate protein ligase A; the E2p LD mutant could be lipoylated only at a significantly lower rate. Likewise, this domain exhibited 1D and 2D NMR spectra characteristic of a partially folded protein, whereas the spectra of mutants with modified loops were similar to those of the wild-type domain. The surface loop is evidently important to the structural integrity of the domain and may help to stabilize the thioester bond linking the acyl group to the reduced lipoyl-lysine swinging arm as part of the catalytic mechanism. Recognition of the lipoyl domain by its partner E1 appears to be a complex process and not attributable to any single determinant on the domain.     

Specificity determinants for the pyruvate dehydrogenase component reaction mapped with mutated and prosthetic group modified lipoyl domains

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys(173)), with three analogs substituted for the lipoyl group on Lys(173), and with selected L1 mutants. Besides Lys(173) mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 A on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. Alignment of over 40 lipoyl domain sequences supports Ser(141), Thr(143), and Glu(179) serving as specificity residues for use by E1 from eukaryotic sources. Extensive interactions of the lipoyl-lysine prosthetic group within the active site are supported by the limited inhibition of E1 acetylation of native L2 by L2 domains altered either by mutation of Lys(173) or enzymatic addition of lipoate analogs to Lys(173). Thus, efficient use by mammalian E1 of cognate lipoyl domains derives from unique surface residues with critical interactions contributed by the universal lipoyl-lysine prosthetic group, key specificity residues, and some conserved residues, particularly Asp(172) adjacent to Lys(173).     

Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented.     

Site-directed mutagenesis and 1H NMR spectroscopy of an interdomain segment in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Deletion of two of the three homologous lipoyl domains that form the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain of the Escherichia coli pyruvate dehydrogenase complex can be achieved by in vitro deletion in the structural gene aceF. A site-directed mutagenesis of this shortened aceF gene was carried out to replace the glutamine residue at position 291 (wild-type numbering) with a histidine residue. Residue 291 is near the middle of a long segment (about 30 amino acid residues) of polypeptide chain, rich in alanine, proline, and charged amino acids, that links the remaining lipoyl domain to the dihydrolipoamide dehydrogenase (E3) binding domain in the E2p chain. A fully active enzyme complex was still assembled, and despite the enormous size of the particle (Mr approximately 4 x 10(6)), sharp resonances attributable to the single new histidine residue per E2p chain could be detected in the 400-MHz 1H NMR spectrum of the complex. The sharpness of these resonances, their chemical shifts (7.94 and 7.05 ppm), and the apparent pKa (6.4) of the side chain were all consistent with this histidine residue being exposed to solvent in a conformationally flexible region of the E2p polypeptide chain. These experiments provide direct proof for the conformational flexibility of this region of polypeptide chain, which is thought to play an important part in the movement of the lipoyl domain required for active site coupling in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex

Reductive acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed specifically by its partner pyruvate decarboxylase (E1p), and no productive interaction occurs with the analogous 2-oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase complex. Residues in the lipoyl-lysine beta-turn region of the unlipoylated E2plip domain (E2plip(apo)) undergo significant changes in both chemical shift and transverse relaxation time (T(2)) in the presence of E1p but not E1o. Residue Gly11, in a prominent surface loop between beta-strands 1 and 2 in the E2plip domain, was also observed to undergo a significant change in chemical shift. Addition of pyruvate to the mixture of E2plip(apo) and E1p caused larger changes in chemical shift and the appearance of multiple cross-peaks for certain residues, suggesting that the domain was experiencing more than one type of interaction. Residues in both beta-strands 4 and 5, together with those in the prominent surface loop and the following beta-strand 2, appeared to be interacting with E1p, as did a small patch of residues centred around Glu31. The values of T(2) across the polypeptide chain backbone were also lower than in the presence of E1p alone, suggesting that E2plip(apo) binds more tightly after the addition of pyruvate. The lipoylated domain (E2plip(holo)) also exhibited significant changes in chemical shift and decreases in the overall T(2) relaxation times in the presence of E1p, the residues principally affected being restricted to the half of the domain that contains the lipoyl-lysine (Lys41) residue. In addition, small chemical shift changes and a general drop in T(2) times in the presence of E1o were observed, indicating that E2plip(holo) can interact, weakly but non-productively, with E1o. It is evident that recognition of the protein domain is the ultimate determinant of whether reductive acetylation of the lipoyl group occurs, and that this is ensured by a mosaic of interactions with the Elp.     

Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus.

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus comprises a structural core, composed of 60 dihydrolipoamide acetyltransferase (E2p) subunits, which binds multiple copies of pyruvate decarboxylase (E1p) and dihydrolipoamide dehydrogenase (E3) subunits. After limited proteolysis with chymotrypsin, the N-terminal lipoyl domain of E2p was excised, purified and sequenced. The residual complex, which remained assembled, was then digested with trypsin under mild conditions. This treatment promoted complete disassembly of the complex and the various components were separated by gel filtration and h.p.l.c. A folded fragment of E2p containing about 50 amino acid residues was identified as being responsible for binding the E3 subunits, although, unlike the corresponding region of the E2p or E2o chains of the pyruvate dehydrogenase or 2-oxoglutarate dehydrogenase complexes from Escherichia coli, the fragment also bound E1p molecules. Further peptide purification and sequence analysis allowed the determination of the first 211 amino acid residues of the B. stearothermophilus E2p chain, thus providing the complete primary structure of the lipoyl domain, the E1p/E3-binding domain and the regions of polypeptide chain, probably highly flexible in nature, that link the domains to each other and to the inner-core (E2p-binding) domain. Several of the proteolytically sensitive sites were also identified. The sequence of the B. stearothermophilus E2p chain shows close homology with the sequences of the E2p and E2o chains from E. coli, although significant differences in structure are apparent. Detailed evidence for the sequence of the peptides obtained by limited proteolysis and further chemical and enzymic cleavages have been deposited as Supplementary Publication SUP 50142 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 6BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.     

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.     

Overexpression of restructured pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue.

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase multienzyme complex (PDH complex). A thermoinducible expression system was developed to amplify a variety of genetically restructured PDH complexes, including those containing three, two, one and no lipoyl domains per E2p chain. Although large quantities of the corresponding complexes were produced, they had only 20-50% of the predicted specific activities. The activities of the E1p components were diminished to the same extent, and this could account for the shortfall in overall complex activity. Thermoinduction was used to express a mutant PDH complex in which the putative active-site histidine residue of the E2p component (His-602) was replaced by cysteine in the H602C E2p component. This substitution abolished dihydrolipoamide acetyltransferase activity of the complex without affecting other E2p functions. The results support the view that His-602 is an active-site residue. The inactivation could mean that the histidine residue performs an essential role in the acetyltransferase reaction mechanism, or that the reaction is blocked by an irreversible modification of the cysteine substituent. Complementation was observed between the H602C PDH complex and a complex that is totally deficient in lipoyl domains, both in vitro, by the restoration of overall complex activity in mixed extracts, and in vivo, from the nutritional independence of strains that co-express the two complexes from different plasmids.     

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.     

The role of loop and beta-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex

The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a beta-turn. Residues within the lipoyl-lysine beta-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the beta-turn residue on the N-terminal side of the E2o lipoyl-lysine DK(A)/(V) motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.     

Restricted motion of the lipoyl-lysine swinging arm in the pyruvate dehydrogenase complex of Escherichia coli

The three lipoyl (E2plip) domains of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase (PDH) complex of Escherichia coli house the lipoyl-lysine side chain essential for active-site coupling and substrate channelling within the complex. The structure of the unlipoylated form of the innermost domain (E2plip(apo)) was determined by multidimensional NMR spectroscopy and found to resemble closely that of a nonfunctional hybrid domain determined previously [Green et al. (1995) J. Mol. Biol. 248, 328-343]. The domain comprises two four-stranded beta-sheets, with the target lysine residue residing at the tip of a type-I beta-turn in one of the sheets; the N- and C-termini lie close together at the opposite end of the molecule in the other beta-sheet. Measurement of (15)N NMR relaxation parameters and backbone hydrogen/deuterium (H/D) exchange rates reveals that the residues in and surrounding the lipoyl-lysine beta-turn in the E2plip(apo) form of the domain become less flexible after lipoylation of the lysine residue. This implies that the lipoyl-lysine side chain may not sample the full range of conformational space once thought. Moreover, reductive acetylation of the lipoylated domain (E2plip(holo) --> E2plip(redac)) was accompanied by large changes in chemical shift between the two forms, and multiple resonances were observed for several residues. This implies a change in conformation and the existence of multiple conformations of the domain on reductive acetylation, which may be important in stabilizing this catalytic intermediate.     

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

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

Repeating functional domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Each polypeptide chain in the lipoate acetyltransferase (E2) core of the pyruvate dehydrogenase complex from Escherichia coli contains three repeating sequences in the N-terminal half of the molecule. The repeats are highly homologous in primary structure and each includes a lysine residue that is a potential site for lipoylation. We have shown that all three sites are lipoylated, at least in part, and that the three lipoylated segments of the E2 chain can be isolated as distinct functional domains after limited proteolysis. Each domain becomes partly acetylated in the intact complex in the presence of substrate. In the primary structure, the domains are separated by regions of polypeptide chain oddly rich in alanine and proline residues. These regions are probably the conformationally mobile segments observed in the 1H-n.m.r. spectrum of the complex and which are removed by tryptic cleavage at Lys-316. The C-terminal half of the molecule contains the acetyltransferase active site and the binding sites for E1, E3 and other E2 subunits. The pyruvate dehydrogenase complex of E. coli, which has a heterogeneous quaternary structure, is thus far unique among the 2-oxo acid dehydrogenase complexes in possessing more than one lipoyl domain per E2 chain, but this may be a general feature of the enzyme from Gram-negative organisms.     

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.