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Global view of cognate kinase activation by the human pyruvate dehydrogenase complex.

ABSTRACT: The human pyruvate dehydrogenase complex (PDC) comprises four multidomain components, E1, E3, E2 and an E3-binding protein (E3BP), the latter two forming the core as E2·E3BP sub-complex. Pyruvate flux through PDC is regulated via phosphorylation (inactivation) at E1 by four PDC kinases (PDKs), and reactivation by two PDC phosphatases. Up-regulation of PDK isoform gene expression is reported in several forms of cancer, while PDKs may be further activated by PDC by binding to the E2·E3BP core. Hence, the PDK: E2·E3BP interaction provides new therapeutic targets. We carried out both functional kinetic and thermodynamic studies to demonstrate significant differences in the activation of PDK isoforms by binding to the E2·E3BP core: (i) PDK2 needs no activation by E2·E3BP for efficient functioning, while PDK4 was the least effective of the four isoforms, and could not be activated by E2·E3BP. Hence, development of inhibitors to the interaction of PDK2 and PDK4 with E2·E3BP is not promising; (ii) Design of inhibitors to interfere with interaction of E2·E3BP with PDK1 and PDK3 is promising. PDK3 needs E2·E3BP core for activation, an activation best achieved by synergistic combination of E2-derived catalytic domain and tridomain.

SUBMITTER: Guevara EL

PROVIDER: S-EPMC5322387 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Global view of cognate kinase activation by the human pyruvate dehydrogenase complex.

Guevara Elena L EL Yang Luying L Birkaya Barbara B Zhou Jieyu J Nemeria Natalia S NS Patel Mulchand S MS Jordan Frank F

Scientific reports 20170223

The human pyruvate dehydrogenase complex (PDC) comprises four multidomain components, E1, E3, E2 and an E3-binding protein (E3BP), the latter two forming the core as E2·E3BP sub-complex. Pyruvate flux through PDC is regulated via phosphorylation (inactivation) at E1 by four PDC kinases (PDKs), and reactivation by two PDC phosphatases. Up-regulation of PDK isoform gene expression is reported in several forms of cancer, while PDKs may be further activated by PDC by binding to the E2·E3BP core. Hen ...[more]

PMID: 28230160

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Project description:Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2+E3BP) core organisation: the 'addition' model (60+12) and the 'substitution' model (48+12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the 'substitution' model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.

Dataset Information

Global view of cognate kinase activation by the human pyruvate dehydrogenase complex.

Publications

Global view of cognate kinase activation by the human pyruvate dehydrogenase complex.

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