title: Pivotal role of the C-terminal DW-motif in mediating inhibition of pyruvate dehydrogenase kinase 2 by dichloroacetate.
abstract: The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20-60% residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC(50) values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DW-motif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DW-motif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Masato
authors.initials: M
authors.lastname: Kato
authors.forename: David T
authors.initials: DT
authors.lastname: Chuang
mesh_terms: D020816:Amino Acid Motifs; D003999:Dichloroacetic Acid / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008968:Molecular Conformation; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D015398:Signal Transduction
chemical_list: D004791:Enzyme Inhibitors; D008055:Lipids; C000631430:PDK2 protein, human; D020033:Protein Isoforms; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D003999:Dichloroacetic Acid; D017346:Protein Serine-Threonine Kinases