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 socalled "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.Oxidative decarboxylation of pyruvate catalyzed by the mitochondrial pyruvate dehydrogenase complex (PDC) 1 serves as an important metabolic link that connects glycolysis and the citric acid cycle. It is generally believed that the pyruvate dehydrogenase reaction is the rate-limiting step in the aerobic stage of oxidation of carbohydrate fuels (1). The activity of PDC is regulated through a reversible phosphorylation (inactivation)-dephosphorylation (reactivation) cycle catalyzed by a dedicated pyruvate dehydrogenase kinase (PDHK) and pyruvate dehydrogenase phosphatase (PDP), respectively (2). Under most circumstances, both PDHK and PDP are continuously active, maintaining a certain phosphorylation state or activity state of PDC. Under normal feeding conditions, the activity state of PDC in different tissues varies on average from † This work was supported by Grant GM51262 from the U.S. Public Health Service.* To whom correspondence should be addressed: Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama, KAUL 440A, 720 20th St. South, Birmingham, 934-0758. E-mail: kpopov@uab.edu. 1 Abbreviations: PDC, pyruvate dehydrogenase complex; PDHK, pyruvate dehydrogenase kinase; PDHK1-PDHK4, isozymes 1-4 of pyruvate dehydrogenase kinase, respectively; E1, pyruvate dehydrogenase component of PDC; E2, dihydrolipoyl transa...

Distinct Structural Mechanisms for Inhibition of Pyruvate Dehydrogenase Kinase Isoforms by AZD7545, Dichloroacetate, and Radicicol

“…In the presence of the E2p/E3BP core, AZD7545 at 10 μM reduces PDK1 and PDK3 activities to 28% and 31% of the control, respectively ( Figure 2C). These data are consistent with an earlier proposition that AZD7545 inhibits the E2p-dependent stimulation of PDK2 activity (Morrell et al, 2003;Tuganova et al, 2007). Paradoxically, when the E2p/E3BP core is absent, AZD7545 stimulates scaffold-free basal PDK1 and PDK3 activities to 1.3-fold and 10-fold, respectively ( Figure 2D).…”

A combination of alpha lipoic acid and calcium hydroxycitrate is efficient against mouse cancer models: Preliminary results

“…Pyruvate dehydrogenase kinase (PDHK) is an integral part of the PDC, with two or three PDHK dimers per complex. Inactivation of the E1 component of the PDC complex occurs when PDHK phosphorylates three serine residues on E1 (ser264, ser271 and ser203) (47). It has been shown that lipoic acid restores the activity of PDH through the inhibition of PDHK (26).…”

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

“…DCA is disadvantageous as a drug because of its low affinity for PDK isoforms (31,43) and potential toxicity to humans (44). A recently developed glucose-lowering compound AZD7545 (2R-N-{4-[4-(dimethylcarbamoyl)phenylsulfonyl]-2-chlorophenyl}-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide) (45) functions as a dihydrolipoamide mimetic (46) and binds to the lipoyl domain-binding pocket in the PDK1-AZD7545 structure (41). Paradoxically, AZD7545 serves as a "dual role" ligand for PDK3.…”