Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2

Abstract

Pyruvate dehydrogenase kinase 2 (PDHK2) is a unique mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase multienzyme complex (PDC). PDHK2 is an integral component of PDC tightly bound to the inner lipoyl-bearing domains (L2) of the dihydrolipoyl transacetylase component (E2) of PDC. This association has been reported to bring about an up to 10-fold increase in kinase activity. Despite the central role played by E2 in the maintenance of PDHK2 functionality in the PDC-bound state, the molecular mechanisms responsible for the recognition of L2 by PDHK2 and for the E2-dependent PDHK2 activation are largely unknown. In this study, we used a combination of molecular modeling and site-directed mutagenesis to identify the amino acid residues essential for the interaction between PDHK2 and L2 and for the activation of PDHK2 by E2. On the basis of the results of site-directed mutagenesis, it appears that a number of PDHK2 residues located in its R domain (P22, L23, F28, F31, F44, L45, and L160) and in the so-called "cross arm" structure (K368, R372, and K391) are critical in determining the strength of the interaction between PDHK2 and L2. The residues of L2 essential for recognition by PDHK2 include L140, K173, I176, E179, and to a lesser extent D164, D172, and A174. Importantly, certain PDHK2 residues forming interfaces with L2, i.e., K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state. Finally, evidence that the blood glucose-lowering compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity is presented.

Figure 1

Recognition of L2 by PDHK2. (A) Space filling representation of the PDHK2 dimer viewed down the lipoyl-bearing domain-binding site. The model of the PDHK2–L2 complex was based on the published structure of the PDHK3–L2 complex [PDB entry 1y8n (36)] using the homology modeling program Swiss-Model (39). PDHK2 subunit I is colored yellow. PDHK2 subunit II is colored green. Amino acid residues of PDHK2 located within contact distance of L2 in the PDHK2–L2 complex are highlighted: basic residues are colored blue, acidic residues red, and neutral residues sand. (B) Space filling representation of the R domain of PDHK2 subunit II viewed down the lipoate-binding cavity. Lipoate residues (LPA) are shown as a stick model. Hydrophobic residues lining up the lipoate-binding cavity are the color of sand. Residues L160, I167, and F168 located inside of the lipoate-binding cavity are not shown. (C) Space filling representation of L2 based on 1y8n coordinates (36). Amino acid residues of L2 located within contact distance of PDHK2 in the PDHK2–L2 complex are highlighted: basic residues are colored blue, acidic residues red, and neutral residues sand. The graphics were generated using PyMOL, version 0.98.

Figure 2

Binding of wild-type and mutant PDHK2 proteins to the unaltered L2. (A) SDS–PAGE analysis of wild-type and mutant PDHK2 proteins pulled down using an unaltered GST-L2 construct. Pulldown experiments were carried out as described previously (27). Gels were stained with Coomassie R250. Shown are representative data from four experiments. (B) Binding isotherms of L2 and PDHK2. ITC measurements were performed at 30 °C in a VP-ITC microcalorimeter (MicroCal). Unaltered L2 (250 μM) in the syringe was injected into the reaction cell containing 10 μM wild-type or mutant PDHK2. The molar ratio represents L2 monomer to PDHK2 dimer. Solid lines depict the least-square fitting curves obtained using a single-site L2 binding model. Fittings were made using Origin, version 7.0. Data for binding of wild-type PDHK2 are shown as empty circles. Data for binding of PDHK2-K17A, PDHK2-Q47A, PDHK2-R372A, and PDHK2-K391A proteins are shown as empty triangles, empty squares, filled squares, and filled triangles, respectively.

Figure 3

Binding of unaltered and mutant L2 proteins to wild-type PDHK2. (A) SDS–PAGE analysis of wild-type PDHK2 pulled down using unaltered and mutant GST-L2 proteins. Pulldown experiments were carried out as described previously (27). Gels were stained with Coomassie R250. Shown are representative data of five experiments. (B) Binding isotherms of L2 and PDHK2. ITC measurements were performed at 30 °C in a VP-ITC microcalorimeter (MicroCal). Unaltered or mutant L2 (250 μM) in the syringe was injected into the reaction cell containing 10 μM wild-type PDHK2. The molar ratio represents L2 monomer to PDHK2 dimer. Solid lines depict the least-square fitting curves obtained using a single-site L2 binding model. Fittings were made using Origin, version 7.0. Data for binding of unaltered L2 are shown as empty circles. Data for binding of L2-E162A, L2-D164A, L2-K173A, and L2-E183A proteins are shown as empty squares, filled squares, filled triangles, and filled circles, respectively.

Figure 4

Effect of AZD7545 on wild-type PDHK2. (A) Effects of AZD7545 on PDHK2 activity determined with free E1 (○) or E2-bound E1 (●) as a substrate. The activity of PDHK2 was determined on the basis of the incorporation of [³²P]phosphate into E1 during 1 min of the reaction as described previously (18). Each data point represents the mean ± the standard deviation for three to five independent determinations. (B) Effect of AZD7545 on binding of PDHK2 to the unaltered GST-L2 protein. The left panel depicts experiments carried out with vehicle alone. The right panel shows the experiment carried out with 6 μM AZD7545 added to the binding mixture. Shown are representative data from four experiments. (C) Displacement of PDHK2 from the PDHK2–GST-L2 complex by AZD7545. Preformed PDHK2–GST-L2 complexes were incubated in binding buffer with vehicle (left) or 6 μM AZD7545 (right). Protein contents of free (F) and bound (B) fractions were analyzed using SDS–PAGE. Gels were stained with Coomassie R250. Shown are representative data from four experiments. AZD7545 was added from the stock solution made in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the binding mixture was less than 1% (v/v).

Figure 5

Effect of AZD7545 on the activity of wild-type and mutant PDHK2. (A) The effect of AZD7545 on the activity of wild-type PDHK2 is shown with filled circles. The effects of AZD7545 on the activities of PDHK2-F31A and PDHK2-F44A are shown with filled rectangles and filled triangles, respectively. The activity of PDHK2 was determined on the basis of the incorporation of [³²P]phosphate into the E2/E3BP-bound E1 subunit. Kinetic data were fitted and analyzed using GraFit, version 5.0. Each data point represents the mean ± the standard deviation for three to five independent determinations. AZD7545 was added from the stock solutions made in DMSO. The final concentration of DMSO in reaction mixtures was 1% (v/v). (B) CD spectra of wild-type PDHK2 (–) and PDHK2-F44A (- - -) proteins. CD spectra were recorded using a Jasco J815 spectrometer (Jasco Inc., Easton, MD) as described in Experimental Procedures.