BINDING OF THE PYRUVATE-DEHYDROGENASE KINASE TO RECOMBINANT CONSTRUCTS CONTAINING THE INNER LIPOYL DOMAIN OF THE DIHYDROLIPOYL ACETYLTRANSFERASE COMPONENT

The dihydrolipoyl acetyltransferase (E2) component of the mammalian py ruvate dehydrogenase complex forms a 60-subunit core in which E2's inn er domain forms a dodecahedron shaped structure surrounded by its glob ular outer domains that are connected to each other and the inner doma in by 2-3-kDa mobile hinge regions. Two of the outer domains are simil ar to 10 kDa lipoyl domains, an NH2-terminal one, E2(L1), and, after t he first hinge region a second one, E2(L2). The pyruvate dehydrogenase kinase binds tightly to the lipoyl domain region of the oligomeric E2 core and phosphorylates and inactivates the pyruvate dehydrogenase (E 1) component. We wished to determine whether lipoyl domain constructs prepared by recombinant techniques from a cDNA for human E2 could bind the bovine E1 kinase and, that being the case, to pursue which lipoyl domain the kinase binds. We also wished to gain insights into how a m olecule of kinase tightly bound to the E2 core can rapidly phosphoryla te 20-30 molecules of the pyruvate dehydrogenase (E1) component which are also bound to an outer domain of the E2 core. We prepared recombin ant constructs consisting of the entire lipoyl domain region or the in dividual lipoyl domains with or without the intervening hinge region. Constructs were made and used both as free lipoyl domains and fused to glutathione S-transferase (GST). Using GSH-Sepharose to selectively b ind GST constructs, tightly bound kinase was shown to rapidly transfer in a highly preferential way from intact E2 core to GST constructs co ntaining the E2(L2) domain rather than to ones containing only the E2( L1) domain. GST E2(L2)-kinase complexes could be eluted from GSH-Sepha rose with glutathione. Delipoylation of E2(L2) by treatment with lipoa midase eliminated kinase binding supporting a direct role of the lipoy l prosthetic group in this association. Transfer to and selective bind ing of the kinase by E2(L2) but not E2(L1) was also demonstrated with free constructs using a sucrose gradient procedure to separate the lar ge E2 core from the various lipoyl domain constructs. E2(L2) but not E 2(L1) increased the activity of resolved kinase by up to 43%. We concl ude that the kinase selectively binds to the inner lipoyl domain of E2 subunits and that this association involves its lipoyl prosthetic gro up. We further suggest that transfer of tightly bound kinase between E 2(L2) domains occurs by a direct interchange mechanism without formati on of free kinase (model presented), Such interdomain movement would e xplain how a kinase molecule can rapidly phosphorylate a large complem ent of pyruvate dehydrogenase tetramers which are bound throughout the surface of the E2 oligomer.