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J. Biol. Chem., Vol. 283, Issue 37, 25305-25315, September 12, 2008

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Pyruvate Dehydrogenase Kinase-4 Structures Reveal a Metastable Open Conformation Fostering Robust Core-free Basal Activity^*

R. Max Wynn, Masato Kato, Jacinta L. Chuang, Shih-Chia Tso, Jun Li, and David T. Chuang¹

From the Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038

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-Å 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 (Asp³⁹⁴–Trp³⁹⁵) 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.

Received for publication, March 21, 2008 , and in revised form, July 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant DK62306. This work was also supported by Grant I-1286 from the Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 2ZKJ and 3D2R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed. E-mail: David.Chuang{at}UTSouthwestern.edu.

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