Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly

The final version of this article is available at the link below.Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1–E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric ‘core’ provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an ‘average’ 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.This work was partly supported by EPSRC (under grants GR/R99393/01 and EP/C015452/1)

Ansatzpunkte einer oekonomischen Theorie konkurrierender Nutzungen von Wasserressourcen

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Data collection and refinement statistics.

<p>*Values in parentheses are for the highest-resolution shell.</p><p>Data collection and refinement statistics.</p

The effect of F190L to peroxidase activity and sensitivity to hyperoxidation.

<p><b>a.</b> Time course of PrxIII dependent NADPH oxidation coupled to H₂O₂ reduction for PrxIII wild-type and F190L. NADPH oxidation was monitored at 340 nm in a 1 ml reaction mixture containing with 5 μM of PrxIII, 2.1 μM Trx, 1.5 μM TrxR2 and 0.5 mM NADPH in 50 mM NaH₂PO4 buffer, pH 7.4 containing 150 mM NaCl, 1 mM EDTA. A reaction mix without PrxIII was used as the negative control. This is a representative result from three independent experiments with similar results. <b>b.</b> Dose-dependent increase in PrxIII wild-type and F190L mutant hyperoxidation after exposure to hydrogen peroxide at the indicated concentrations. PrxIII hyper-oxidation was monitored by western blotting using an antibody that recognizes primarily the sulfinic or sulfonic acid forms of Prxs (α-SO_2-3). The assay was carried out in the presence of 1 mM DTT as reductant. Western blots were used for quantification of hyperoxidized PrxIII by densitometry. Intensity of hyper-oxidized PrxIII was normalized against loading control blotted with an anti-His antibody. Error bars represent the mean ±S.D. from three independent experiments.</p

Arg123 and the Arg-Glu-Arg network are in a new conformational state.

<p><b>a.</b> Structure alignment between reduced PrxIII F190L (orange) and PrxIV C51A (cyan) showing Arg123, Glu50 and Arg146 are in different positions. The position of the peroxidatic cysteine is fixed. The 2Fo-Fc electron density map around the residues of PrxIII is contoured at 1.0 σ. <b>b.</b> Detailed representation of the hydrogen bonding network in both reduced and oxidized PrxIII and PrxIV from the same viewpoint. Hydrogen bonds are shown in dotted yellow lines.</p

PrxIII forms interlocked rings in both oxidized and states.

<p>a. Detailed cartoon diagram of reduced and oxidized PrxIII F190L showing the transition between fully folded (FF) and locally unfolded (LU) forms. The active-site cysteine sulfur atom is shown as yellow sticks. Different chains are colored in cyan and blue, respectively. b. Surface diagram of the reduced PrxIII F190L catenane showing two interlocking dodecameric rings in orange and blue, respectively. Both oxidized and reduced states of the protein display a very similar catenaned structure.</p

Ring-ring interactions.

<p>Detailed representation of the hydrogen bonding network in different contact areas between the rings. The two rings are colored in blue and orange, respectively. The hydrogen bonds are shown in dotted yellow lines with the distance in angstroms (Å).</p

Schematic illustration of various states of PrxIII during the reaction cycle.

<p>The homodimer of the PrxIII dodecamer represents a functional unit during the reaction cycle: (i) the peroxidatic cysteine (SH_per) reduces hydrogen peroxide and is converted to its sulfenylated (SOH) form. (ii) the Cp loop housing the peroxidatic cysteine unfolds from its FF to LU conformation. (iii) the peroxidatic cysteine forms a disulfide bond with the resolving cysteine eliminating an H₂O molecule (iv) mitochondrial thioredoxin (Trx_red) reduces the disulfide bond to regenerate the reduced active-site cysteine while the Cp loop re-assumes the FF state conformation. The sulfenylated cysteine intermediate can be further oxidized to its sulfinylated or sulphonylated forms while it remains in the FF state at elevated H₂O₂ levels.</p