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.

Figures

FIGURE 1.

Crystal structure of human PDK2 with different bound ligands and the DW-motif anchoring site. A, ligand-binding sites in the N-terminal domain of human PDK2. The structure of human PDK2 bound to ADP and DCA (Protein Data Bank code 2BU8) (21) was superimposed on that of human PDK1 in complex with AZD7545 (Protein Data Bank code 2Q8G) (22), and only AZD7545 was shown in the PDK2 structure. The N-terminal domain (green) of PDK2 is shown in front of the C-terminal domain. The bound ligands, ADP, DCA, and AZD7545, are shown as sphere models. The residues involved in ligand binding and putative communicating residues between the ligand-binding sites are shown as stick models. The ATP lid is shown in yellow, and the DW-motif in the C-terminal tail from subunit II in the PDK2 dimer is shown in magenta. For clarity, α-helix 5 of PDK2 is removed from the figure. B, close-up view of the DW-motif anchoring site (adapted from 39). The side chains of Asp-382′ and Trp-383′ (DW, in magenta) and the interacting residues in the N-terminal domain (green) are shown as stick models. The dashed lines in magenta indicate disordered regions upstream and downstream of the DW-motif. The gray dashed lines indicate hydrogen bonds. The cation-π interaction between the side chain of Try-383 and two arginine residues (Arg-149 and Arg-362) are represented by dotted spheres. Molecular graphics and superimpositions of the structures were carried out using PyMOL (DeLano Scientific, LLC, Palo Alto, CA).

FIGURE 2.

Kinase activities in wild-type and variant PDK2. The phosphorylation reaction was carried out in the absence and presence of the 60-meric E2p/E3BP core as described under “Experimental Procedures.” The specific activities were expressed as nmol of 32P incorporated/min/mg of kinase. Bars, averages ± S.D. (n = 3–5).

FIGURE 3.

Inhibition of PDK2 basal and E2p/E3BP-dependent kinase activities by DCA. A, inhibition of PDK2 core-free basal activities (without E2/E3BP) by DCA (in a concentration range from 0.1 μm to 500 mm), which is expressed as percentage of control activities (measured without DCA). The phosphorylation reaction was conducted essentially as described in the legend to Fig. 2 except for the inclusion of DCA at different concentrations. Each point represents the average of two independent reactions. B, inhibition of PDK2 core-dependent activities (with 60-meric E2/E3BP) by DCA (in a concentration range from 0.1 μm to 500 mm), expressed as percentage of control activities (measured without DCA). All data were plotted with the GraphPad Prism program.

FIGURE 4.

Tryptophan fluorescence quenching in wild-type and mutant PDK2. Tryptophan fluorescence quenching from the DW-motif was monitored by recording emission spectra (averaged from three consecutive scans) between 320 and 420 nm in a Fluorolog 3 spectrofluorometer (HORIBA Jobin Yvon, Edison, NJ), with the excitation wavelength set at 290 nm. Initial tag-free PDK2 concentrations (based on the dimer) were 0.5 μm in 50 mm potassium phosphate buffer (pH 7.5) containing 0.2 mm EDTA and 2 mm MgCl2. A, fluorescence spectra of WT and mutant PDK2 in the absence of DCA or ADP. B, quenching of PDK2 fluorescence by increasing concentrations of DCA. C, quenching of PDK2 fluorescence by 50 μm ADP and increasing concentrations of DCA. The quenching data were fit as described (39) and plotted with the GraphPad Prism program.

FIGURE 5.

Distinct binding properties of lipL2 to wild-type and mutant PDK2 in the absence or presence of different ligands measured by ITC. Binding affinities of wild-type and mutant PDK2 for lipL2 were determined by injecting 500 μm lipL2 in 10-μl increments into the cell containing 1.8 ml of 30 μm PDK2 (based on the monomer). Ligands (ADP, DCA, or ADP and DCA), alone or in combination, were included in both the syringe and the cell in equal concentrations. Titration data were initially processed by the Origin 7 program and subsequently presented with the GraphPad Prism program. A, WT-PDK2, WT-PDK2 in 0.3 mm DCA, WT-PDK2 in 0.1 mm ADP, WT-PDK2 in 0.3 mm DCA, and 0.1 mm ADP were titrated with lipL2 in the absence or presence of corresponding ligands. B, Y145F-PDK2, Y145F in 0.3 mm DCA, Y145F-PDK2 in 0.1 mm ADP, and R149A-PDK2 were titrated in the absence or presence of the corresponding ligands.

FIGURE 6.

Binding of AZD7545 to wild-type and mutant PDK2 in the absence or presence of different ligands measured by ITC. Binding affinities of PDK2 and AZD7545 were determined by injecting 100 μm AZD7545 in 10-μl increments into the cell containing 1.8 ml of 10 μm PDK2 (based on the monomer). Ligands (DCA and ATP or ADP) were included in both the syringe and the cell in equal concentrations. Titration data were initially processed by the Origin 7 program and subsequently presented with the GraphPad Prism program.

FIGURE 7.

The signaling network between different ligand-binding sites in human PDK2. This figure originates from Fig. 1A with minor modifications. As a signal axis, helices α6, α7, and α8 are highlighted as solid schematic models with the rest of the protein shown as a transparency. The red arrows indicate the directional communications between the different ligand binding sites. See “Discussion” for details of site-to-site communications mediated by the DW-motif.

FIGURE 8.

Conformational changes in the lipoyl domain-binding site induced by DCA and ADP binding. A, a steric clash between AZD7545 and the loop region at the lipoyl-binding site of PDK2. The structures of apo-PDK2 (green, Protein Data Bank code 2BTZ) (21) and PDK2-ADP-DCA (Protein Data Bank code 2BU8 magenta) are superimposed on that of PDK1-AZD7545 (orange, Protein Data Bank code 2Q8G) (22). PDK residue numbers include the leader sequence. Asn-35 in the flipped loop between helices α2 and α3 of PDK2-ADP-DCA could cause a steric clash with the bound AZD7545 in PDK1, which may explain the failure of WT PDK2 to bind AZD7545 in the presence of both ADP and DCA (Table 5). AZD7545 is shown as a solid sphere model. The van der Waals radius of Asn-35 is depicted with dotted spheres. B, comparison of the lipoyl-binding site in the PDK2-L2 and PDK2-ADP-DCA structures. The PDK2-L2 complex structure (Protein Data Bank code 3CRL) (48) (PDK2 in blue and L2 in yellow) is superimposed on the structure of the PDK2-ADP-DCA ternary complex (21) in magenta. The ordered C-terminal tail in the PDK2-L2 is shown in cyan. The inversion of the loop between helices α2 and α3 induced by ADP and DCA binding would not interfere with the binding of lipoyl group of L2 to PDK2.