Mechanism and Interactions of Human Pyruvate Dehydrogenase Complex with its Kinase1

The human pyruvate dehydrogenase complex (PDC) consist of three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3‐binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1–4), and inactivation reversed by the action of two phosphatases (PDP1 and 2). Specifically, the regulation of PDC via phosphorylation of E1 by PDK1 is the focus of these studies, as PDK1 has been strongly implicated as an overexpressed culprit in multiple cancer cells; hence inhibition of the interaction of PDK1 with the E1 or E2•E3BP, could provide novel targets for the design of anticancer agents. Since the PDKs reside on (have a stronger binding to) the E2•E3BP core of the entire complex, specific inhibition of the PDK1‐E2•E3BP interaction provides a possible new approach to treatment of cancer. Inactivation of E1 via phosphorylation by PDK1‐4 was initially studied to determine PDK interaction and therefore activation by E2•E3BP or its C‐terminally truncated proteins. The studies suggest that the individual PDK isoforms require different degrees of activation: PDK2 appears to be the most active and not highly selective whereas PDK4 is the least active never fully inactivating E1. Recently, the interaction loci between PDK1 (PDK2) and the E2•E3BP core was defined by the Jordan group by two complementary methods, HD‐exchange MS (HDX‐MS) and NMR1. Interrogation of the sites on E2•E3BP identified to interact with PDK1 (PDK2) was accomplished by site‐directed mutagenesis studies in order to validate the identified ‘hot spots’. To date, there is evidence of the variants on the L1L2S region of E2 effecting inactivation of E1 via phosphorylation by PDK1. Further studies will be performed to identify the protein‐protein interaction of these L1L2S variants with PDK1 and PDK2 using HDX‐MS, isothermal titration calorimetry (ITC) and fluorescence titrations. These measurements will confirm whether the ‘hot spots’ interrogated indeed are important for the interaction of E2•E3BP with the PDK1, and whether it is specific to PDK1 by comparing such measurements with PDK2. These experiments will identify ‘real’ hot spots, against which rational drug design could be undertaken. Support or Funding Information This research was supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award GM113601‐01A1 from the National Institute of General Medical Sciences.