A metabolite binding protein moonlights as a bile-responsive chaperone.

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.

Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy.

In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR.

The trans-to-cis proline isomerization in E. coli Trx folding is accelerated by trans prolines.

The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coli Trx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coli Trx contains four additional prolines, which are all in the trans conformation in the native state. Notably, a recent study revealed that replacement of all four trans prolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four trans prolines accelerate the trans-to-cis transition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural trans prolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cis isomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that trans prolines can significantly modulate the kinetics of rate-limiting trans-to-cis proline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.

Donor strand sequence, rather than donor strand orientation, determines the stability and non-equilibrium folding of the type 1 pilus subunit FimA.

FimA is the main structural subunit of adhesive type 1 pili from uropathogenic Escherichia coli strains. Up to 3000 copies of FimA assemble to the helical pilus rod through a mechanism termed donor strand complementation, in which the incomplete immunoglobulin-like fold of each FimA subunit is complemented by the N-terminal extension (Nte) of the next subunit. The Nte of FimA, which exhibits a pseudo-palindromic sequence, is inserted in an antiparallel orientation relative to the last ß-strand of the preceding subunit in the pilus. The resulting subunit-subunit interactions are extraordinarily stable against dissociation and unfolding. Alternatively, FimA can fold to a self-complemented monomer with anti-apoptotic activity, in which the Nte inserts intramolecularly into the FimA core in the opposite, parallel orientation. The FimA monomers, however, show dramatically lower thermodynamic stability compared with FimA subunits in the assembled pilus. Using self-complemented FimA variants with reversed, pseudo-palindromic extensions, we demonstrate that the high stability of FimA polymers is primarily caused by the specific interactions between the side chains of the Nte residues and the FimA core and not by the antiparallel orientation of the donor strand alone. In addition, we demonstrate that nonequilibrium two-state folding, a hallmark of FimA with the Nte inserted in the pilus rod-like, antiparallel orientation, only depends on the identity of the inserted Nte side chains and not on Nte orientation.

Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria.

Adhesive type 1 pili from enteroinvasive, Gram-negative bacteria mediate attachment to host cells. Up to 3000 copies of the main pilus subunit, FimA, assemble into the filamentous, helical quaternary structure of the pilus rod via a mechanism termed donor-strand complementation, in which the N-terminal extension of each subunit, the donor strand, is inserted into the incomplete immunoglobulin-like fold of the preceding FimA subunit. For FimA from Escherichia coli, it has been previously shown that the protein can also adopt a monomeric, self-complemented conformation in which the donor strand is inserted intramolecularly in the opposite orientation relative to that observed for FimA polymers. Notably, soluble FimA monomers can act as apoptosis inhibitors in epithelial cells after uptake of type 1-piliated pathogens. Here, we show that the FimA orthologues from Escherichia coli, Shigella flexneri, and Salmonella enterica can all fold to form self-complemented monomers. We solved X-ray structures of all three FimA monomers at 0.89-1.69 Å resolutions, revealing identical, intramolecular donor-strand complementation mechanisms. Our results also showed that the pseudo-palindromic sequences of the donor strands in all FimA proteins permit their alternative folding possibilities. All FimA monomers proved to be 50-60 kJ/mol less stable against unfolding than their pilus rod-like counterparts (which exhibited very high energy barriers of unfolding and refolding). We conclude that the ability of FimA to adopt an alternative, monomeric state with anti-apoptotic activity is a general feature of FimA proteins of type 1-piliated bacteria.

Functional analyses of ancestral thioredoxins provide insights into their evolutionary history.

Thioredoxin (Trx) is a conserved, cytosolic reductase in all known organisms. The enzyme receives two electrons from NADPH via thioredoxin reductase (TrxR) and passes them on to multiple cellular reductases via disulfide exchange. Despite the ubiquity of thioredoxins in all taxa, little is known about the functions of resurrected ancestral thioredoxins in the context of a modern mesophilic organism. Here, we report on functional in vitro and in vivo analyses of seven resurrected Precambrian thioredoxins, dating back 1-4 billion years, in the Escherichia coli cytoplasm. Using synthetic gene constructs for recombinant expression of the ancestral enzymes, along with thermodynamic and kinetic assays, we show that all ancestral thioredoxins, as today's thioredoxins, exhibit strongly reducing redox potentials, suggesting that thioredoxins served as catalysts of cellular reduction reactions from the beginning of evolution, even before the oxygen catastrophe. A detailed, quantitative characterization of their interactions with the electron donor TrxR from Escherichia coli and the electron acceptor methionine sulfoxide reductase, also from E. coli, strongly hinted that thioredoxins and thioredoxin reductases co-evolved and that the promiscuity of thioredoxins toward downstream electron acceptors was maintained during evolution. In summary, our findings suggest that thioredoxins evolved high specificity for their sole electron donor TrxR while maintaining promiscuity to their multiple electron acceptors.

Development of the Mitochondrial Intermembrane Space Disulfide Relay Represents a Critical Step in Eukaryotic Evolution.

The mitochondrial intermembrane space evolved from the bacterial periplasm. Presumably as a consequence of their common origin, most proteins of these compartments are stabilized by structural disulfide bonds. The molecular machineries that mediate oxidative protein folding in bacteria and mitochondria, however, appear to share no common ancestry. Here we tested whether the enzymes Erv1 and Mia40 of the yeast mitochondrial disulfide relay could be functionally replaced by corresponding components of other compartments. We found that the sulfhydryl oxidase Erv1 could be replaced by the Ero1 oxidase or the protein disulfide isomerase from the endoplasmic reticulum, however at the cost of respiration deficiency. In contrast to Erv1, the mitochondrial oxidoreductase Mia40 proved to be indispensable and could not be replaced by thioredoxin-like enzymes, including the cytoplasmic reductase thioredoxin, the periplasmic dithiol oxidase DsbA, and Pdi1. From our studies we conclude that the profound inertness against glutathione, its slow oxidation kinetics and its high affinity to substrates renders Mia40 a unique and essential component of mitochondrial biogenesis. Evidently, the development of a specific mitochondrial disulfide relay system represented a crucial step in the evolution of the eukaryotic cell.

Binding of the Bacterial Adhesin FimH to Its Natural, Multivalent High-Mannose Type Glycan Targets.

Multivalent carbohydrate-lectin interactions at host-pathogen interfaces play a crucial role in the establishment of infections. Although competitive antagonists that prevent pathogen adhesion are promising antimicrobial drugs, the molecular mechanisms underlying these complex adhesion processes are still poorly understood. Here, we characterize the interactions between the fimbrial adhesin FimH from uropathogenic Escherichia coli strains and its natural high-mannose type N-glycan binding epitopes on uroepithelial glycoproteins. Crystal structures and a detailed kinetic characterization of ligand-binding and dissociation revealed that the binding pocket of FimH evolved such that it recognizes the terminal α(1-2)-, α(1-3)-, and α(1-6)-linked mannosides of natural high-mannose type N-glycans with similar affinity. We demonstrate that the 2000-fold higher affinity of the domain-separated state of FimH compared to its domain-associated state is ligand-independent and consistent with a thermodynamic cycle in which ligand-binding shifts the association equilibrium between the FimH lectin and the FimH pilin domain. Moreover, we show that a single N-glycan can bind up to three molecules of FimH, albeit with negative cooperativity, so that a molar excess of accessible N-glycans over FimH on the cell surface favors monovalent FimH binding. Our data provide pivotal insights into the adhesion properties of uropathogenic Escherichia coli strains to their target receptors and a solid basis for the development of effective FimH antagonists.

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.

The Redox State Regulates the Conformation of Rv2466c to Activate the Antitubercular Prodrug TP053.

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.

Accelerating the Association of the Most Stable Protein-Ligand Complex by More than Two Orders of Magnitude.

The complex between the bacterial type 1 pilus subunit FimG and the peptide corresponding to the N-terminal extension (termed donor strand, Ds) of the partner subunit FimF (DsF) shows the strongest reported noncovalent molecular interaction, with a dissociation constant (KD ) of 1.5×10(-20) m. However, the complex only exhibits a slow association rate of 330 m(-1) s(-1) that limits technical applications, such as its use in affinity purification. Herein, a structure-based approach was used to design pairs of FimGt (a FimG variant lacking its own N-terminal extension) and DsF variants with enhanced electrostatic surface complementarity. Association of the best mutant FimGt/DsF pairs was accelerated by more than two orders of magnitude, while the dissociation rates and 3D structures of the improved complexes remained essentially unperturbed. A KD  value of 8.8×10(-22) m was obtained for the best mutant complex, which is the lowest value reported to date for a protein/ligand complex.

How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.

Two critical cysteine residues in the copper-A site (Cu(A)) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu(2+) transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E'0 = -231 mV) as compared with that of TlpA (E'0 = -256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10(4) M(-1) s(-1) that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu(A)-CoxB complex may be bypassed in vivo by high environmental Cu(2+) concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.

Acceleration of the Rate-Limiting Step of Thioredoxin Folding by Replacement of its Conserved cis-Proline with (4 S)-Fluoroproline.

The incorporation of the non-natural amino acids (4R)- and (4S)-fluoroproline (Flp) has been successfully used to improve protein stability, but little is known about their effect on protein folding kinetics. Here we analyzed the influence of (4R)- and (4S)-Flp on the rate-limiting trans-to-cis isomerization of the Ile75-Pro76 peptide bond in the folding of Escherichia coli thioredoxin (Trx). While (4R)-Flp at position 76 had essentially no effect on the isomerization rate in the context of the intact tertiary structure, (4S)-Flp accelerated the folding reaction ninefold. Similarly, tenfold faster trans-to-cis isomerization of Ile75-(4S)-Flp76 relative to Ile75-Pro76 was observed in the unfolded state of Trx. Our results show that the replacement of cis prolines by non-natural proline analogues can be used for modulating the folding rates of proteins with cis prolyl-peptide bonds in the native state.

The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism.

Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores. Among the group of bacterial PFTs are some of the most dangerous toxins, such as diphtheria and anthrax toxin. Examples of eukaryotic PFTs are perforin and the membrane-attack complex, proteins of the immune system. PFTs can be subdivided into two classes, alpha-PFTs and beta-PFTs, depending on the suspected mode of membrane integration, either by alpha-helical or beta-sheet elements. The only high-resolution structure of a transmembrane PFT pore is available for a beta-PFT--alpha-haemolysin from Staphylococcus aureus. Cytolysin A (ClyA, also known as HlyE), an alpha-PFT, is a cytolytic -helical toxin responsible for the haemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is cytotoxic towards cultured mammalian cells, induces apoptosis of macrophages and promotes tissue pervasion. Electron microscopic reconstructions demonstrated that the soluble monomer of ClyA must undergo large conformational changes to form the transmembrane pore. Here we report the 3.3 A crystal structure of the 400 kDa dodecameric transmembrane pore formed by ClyA. The tertiary structure of ClyA protomers in the pore is substantially different from that in the soluble monomer. The conversion involves more than half of all residues. It results in large rearrangements, up to 140 A, of parts of the monomer, reorganization of the hydrophobic core, and transitions of -sheets and loop regions to -helices. The large extent of interdependent conformational changes indicates a sequential mechanism for membrane insertion and pore formation.

Atomic-resolution three-dimensional structure of amyloid ß fibrils bearing the Osaka mutation.

Despite its central importance for understanding the molecular basis of Alzheimer's disease (AD), high-resolution structural information on amyloid ß-peptide (Aß) fibrils, which are intimately linked with AD, is scarce. We report an atomic-resolution fibril structure of the Aß1-40 peptide with the Osaka mutation (E22Δ), associated with early-onset AD. The structure, which differs substantially from all previously proposed models, is based on a large number of unambiguous intra- and intermolecular solid-state NMR distance restraints.

Structural and mechanistic insights into the PAPS-independent sulfotransfer catalyzed by bacterial aryl sulfotransferase and the role of the DsbL/Dsbl system in its folding.

Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.

Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is a soluble, 34 kDa monomer that assembles into a dodecameric pore complex in the presence of membranes or detergent. The comparison of the X-ray structures of monomeric ClyA and the ClyA protomer in the pore complex revealed one of the largest conformational transitions observed so far in proteins, involving the structural rearrangement of more than half of all residues, which is consistent with the finding that conversion from the monomer to the assembly competent protomer is rate-limiting for pore assembly. Here, we introduced artificial disulfide bonds at two distinct sites into the ClyA monomer that both prevent a specific structural rearrangement required for protomer formation. Using electron microscopy and hemolytic activity assays, we show that the engineered disulfides indeed trap these ClyA variants in an assembly incompetent state. Assembly of the variants into functional pore complexes can be completely recovered by disulfide reduction. The assembly kinetics of the ClyA variants recorded with circular dichroism and fluorescence spectroscopy revealed the same mechanism of protomer formation that was observed for wild-type ClyA, proceeding via an intermediate with decreased secondary structure content.

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC.

Type 1 pili from uropathogenic Escherichia coli are filamentous, noncovalent protein complexes mediating bacterial adhesion to the host tissue. All structural pilus subunits are homologous proteins sharing an invariant disulfide bridge. Here we show that disulfide bond formation in the unfolded subunits, catalyzed by the periplasmic oxidoreductase DsbA, is required for subunit recognition by the assembly chaperone FimC and for FimC-catalyzed subunit folding. FimC thus guarantees quantitative disulfide bond formation in each of the up to 3,000 subunits of the pilus. The X-ray structure of the complex between FimC and the main pilus subunit FimA and the kinetics of FimC-catalyzed FimA folding indicate that FimC accelerates folding of pilus subunits by lowering their topological complexity. The kinetic data, together with the measured in vivo concentrations of DsbA and FimC, predict an in vivo half-life of 2 s for oxidative folding of FimA in the periplasm.

Direct evidence for self-propagation of different amyloid-ß fibril conformations.

BACKGROUND: Amyloid fibrils formed by amyloid-ß (Aß) peptides are associated with Alzheimer's disease and can occur in a range of distinct morphologies that are not uniquely determined by the Aß sequence. Whether distinct conformations of Aß fibrils can be stably propagated over multiple cycles of seeding and fibril growth has not been established experimentally. OBJECTIVE: The ability of the 40-residue peptide Aß1-40 to assemble into fibrils with the conformation of the mutant Aß1-40 peptide containing the 'Osaka' mutation E22Δ was investigated. METHODS: Fibril formation of highly pure, recombinant Aß1-40 in the presence of distinct, preformed seeds in vitro was recorded with thioflavin T fluorescence, and distinct fibrillar structures were identified and distinguished by fluorescence spectroscopy and electron microscopy. RESULTS: We propagated the specific quaternary structure of Aß1-40 E22Δ fibrils with wild-type Aß1-40 over up to seven cycles of seeding and fibril elongation. As a result of a 10(7)-fold dilution of the initially present Aß1-40 E22Δ seeds, the vast majority of fibrils formed after the seventh propagation cycle with Aß1-40 did not contain a single molecule of Aß1-40 E22Δ, but still retained the conformation of the initial Aß1-40 E22Δ seeds. Increased critical concentrations of Aß1-40 fibrils formed in the presence of Aß1-40 E22Δ nuclei suggest that these fibrils are less stable than homologously seeded Aß1-40 fibrils, consistent with a kinetically controlled mechanism of fibril formation. CONCLUSION: The propagation of a distinct Aß fibril conformation over multiple cycles of seeded fibril growth demonstrates the basic ability of the Aß peptide to form amyloid strains that in turn may cause phenotypes in Alzheimer's disease.

Dimerization of a 5-kDa domain defines the architecture of the 5-MDa gammaproteobacterial pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E23), we show that two of the three PSBDs in the E23 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E23. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.