Elucidation of the Interaction Loci of the Human Pyruvate Dehydrogenase Complex E2·E3BP Core with Pyruvate Dehydrogenase Kinase 1 and Kinase 2 by H/D Exchange Mass Spectrometry and NMR.
Elucidation of the Interaction Loci of the Human Pyruvate Dehydrogenase Complex E2·E3BP Core with Pyruvate Dehydrogenase Kinase 1 and Kinase 2 by H/D Exchange Mass Spectrometry and NMR.
Biochemistry. 2014 Dec 1;
Authors: Wang J, Kumaran S, Zhou J, Tao H, Nemeria NS, Kakalis L, Patel MS, Park YH, Birkaya B, Jordan F
Abstract
The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2 and E3. The core of the complex is a strong sub-complex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1-4), an inactivation reversed by the action of two phosphatases (PDP1-2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2.E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (LD1 and LD2 on E2 and LD3 on E3BP) lend themselves to NMR studies and determination of interaction maps with the PDK1 and PDK2 at the individual residue level, HDX-MS enabled studies of interaction loci on both partners in the complexes, PDKs and other regions of the E2.E3BP as well, at the peptide level. The HDX-MS suggested that intact E2.E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2.E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence for greater plasticity of PDK1 than PDK2. The effect of L1L2S paralleled HDX-MS results obtained with intact E2.E3BP, hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S' induced moderate interaction with both PDKs according to both methods.
PMID: 25436986 [PubMed - as supplied by publisher]
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