Abstract
Human pyruvate dehydrogenase complex (PDC) is down-regulated by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. PDK4 is overexpressed in skeletal muscle in type 2 diabetes, resulting in impaired glucose utilization. Here we show that human PDK4 has robust core-free basal activity, which is considerably higher than activity levels of other PDK isoforms stimulated by the PDC core. PDK4 binds the L3 lipoyl domain, but its activity is not significantly stimulated by any individual lipoyl domains or the core of PDC. The 2.0-A crystal structures of the PDK4 dimer with bound ADP reveal an open conformation with a wider active-site cleft, compared with that in the closed conformation epitomized by the PDK2-ADP structure. The open conformation in PDK4 shows partially ordered C-terminal cross-tails, in which the conserved DW (Asp(394)-Trp(395)) motif from one subunit anchors to the N-terminal domain of the other subunit. The open conformation fosters a reduced binding affinity for ADP, facilitating the efficient removal of product inhibition by this nucleotide. Alteration or deletion of the DW-motif disrupts the C-terminal cross-tail anchor, resulting in the closed conformation and the nearly complete inactivation of PDK4. Fluorescence quenching and enzyme activity data suggest that compounds AZD7545 and dichloroacetate lock PDK4 in the open and the closed conformational states, respectively. We propose that PDK4 with bound ADP exists in equilibrium between the open and the closed conformations. The favored metastable open conformation is responsible for the robust basal activity of PDK4 in the absence of the PDC core.
Highlights
The human pyruvate dehydrogenase complex (PDC) is primarily regulated by reversible phosphorylation through pyruvate dehydrogenase kinase (PDK) [6, 7]
PDK3 binds to L2 most tightly among the four PDK isoforms and is robustly activated by the E2p/E3-binding protein (E3BP) core; this activation is largely achieved through binding to isolated L2 [17, 18]
Crystal structures of the PDK3-L2-nucleotide complexes revealed that L2 binding to the lipoyl-binding pocket in the N-terminal domain of PDK3 caused an open conformation with a wider active-site cleft in PDK3 [23], compared with the closed conformation present in the L2-free rat PDK2-ADP structure [21, 23]
Summary
Reagents and Recombinant Proteins—Compound AZD7545 [45] was a generous gift from Dr Rachel Mayers, Astrazeneca, UK. The eluted SUMO-PDK4 was further purified on a Superdex 200 column equilibrated with 50 mM potassium phosphate (pH 7.5), 200 mM KCl, 5% glycerol, and 20 mM BME; octyl -D-glucopyranoside was added to peak fractions to a final concentration of 0.05%. Binding Studies by Isothermal Titration Calorimetry—The isolated lipoyl domain (L1, L2, or L3) or the E2p/E3BP core, along with MBP-PDK3 or His tagged PDK4, were dialyzed exhaustively against a buffer containing 50 mM potassium phosphate (K-Pi) (pH 7.5) and 50 mM KCl. For measuring binding of the lipoyl domain to PDK4, the solution of 500 M L1, L2, or L3 in a syringe was injected in 8-l increments into the reaction cell containing 1.8 ml of 80 M PDK4 (based on the monomer) at 20 °C in a VP-ITC microcalorimeter (MicroCal, Northampton, MA). The detailed data processing and curve-fitting procedures for %Qmax and L0.5 have been described previously [56]
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