Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2.
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.
Tuganova
Alina
A
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama, Birmingham, Alabama 35294-0024, USA.
Klyuyeva
Alla
A
Popov
Kirill M
KM
D000813:Anilides / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D051047:Dihydrolipoyllysine-Residue Acetyltransferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051381:Rats; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
C523124:AZD 7545; D000813:Anilides; D007004:Hypoglycemic Agents; D047428:Protein Kinase Inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D008063:Thioctic Acid; C013091:lipoamide; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D017346:Protein Serine-Threonine Kinases