Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly

Vijayakrishnan, S., Kelly, S.M. , Gilbert, R.J.C., Callow, P., Bhella, D. , Forsyth, T., Lindsay, J.G. and Byron, O. (2010) Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly. Journal of Molecular Biology, 399(1), pp. 71-93. (doi:10.1016/j.jmb.2010.03.043) (PMID:20361979) (PMCID:PMC2880790)

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Abstract

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 (E2á+áE3BP) core organisation: the [`]addition' model (60á+á12) and the [`]substitution' model (48á+á12). 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

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Bhella, Dr David and Byron, Professor Olwyn and Vijayakrishnan, Dr Swetha and Lindsay, Professor Gordon and Kelly, Dr Sharon
Authors: Vijayakrishnan, S., Kelly, S.M., Gilbert, R.J.C., Callow, P., Bhella, D., Forsyth, T., Lindsay, J.G., and Byron, O.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > School of Life Sciences
Journal Name:Journal of Molecular Biology
Publisher:Academic Press
ISSN:0022-2836
ISSN (Online):1089-8638
Published Online:31 March 2010
Copyright Holders:Copyright © 2010 Elsevier Ltd.
First Published:First published in Journal of Molecular Biology 399(1):71-93
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
656541Structural studies of human viruses and host interactionsDavid BhellaMedical Research Council (MRC)MC_UU_12014/7MVLS III - CENTRE FOR VIRUS RESEARCH