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The architectural complexity of the human PDC core assembly

Vijayakrishnan, Swetha (2009) The architectural complexity of the human PDC core assembly. PhD thesis, University of Glasgow.

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Abstract

The mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly linking the glycolytic pathway to the TCA cycle via the specific conversion of pyruvate to acetyl CoA and, as such, is responsible for the maintenance of glucose homeostasis in humans. PDC comprises a central pentagonal dodecahedral core of 60 dihydrolipoamide acetyltransferase (E2) and 12 E3 binding protein (E3BP) subunits. Presently, two conflicting models of PDC (E2+E3BP) core organisation exist: the ‘addition’ (60+12) and ‘substitution’ (48+12) models. In addition to its catalytic role, the multi-domain E2/E3BP core provides the structural framework to which 30 pyruvate decarboxylase (E1) heterotetramers and 6-12 dihydrolipoamide dehydrogenase (E3) homodimers are proposed to bind at maximal occupancy. The formation of specific E2:E1 and E3BP:E3 subcomplexes are characteristic of eukaryotic PDCs and are critical for normal complex function. Despite the availability of limited structural data, the exact subunit organisation and mechanism of operation of the mammalian E2/E3BP core remains unknown. This thesis describes the large-scale purification of tagged, recombinant human PDC cores, full-length rE2 and rE2/E3BP, truncated E2/E3BP, peripheral rE3 enzyme as well as native E2/E3BP core (bE2/E3BP) purified from bovine heart. The ability to purify large amounts of pure protein has enabled the characterisation of the individual cores as well as the E2/E3BP:E3 complex using a variety of biochemical and biophysical techniques. Full-length rE2/E3BP, rE2, bE2/E3BP, truncated E2/E3BP (tLi19/tLi30) and rE2/E3BP:E3 were analysed in solution by analytical ultracentrifugation (AUC). While AUC of the cores supported the substitution model of core organisation, the stoichiometry of interaction was determined to be 2:1 (rE2/E3BP:E3). This was further complemented by gel filtration chromatography (GFC) and small angle neutron scattering (SANS), implying the possible existence of a network of E3 ‘cross-bridges’ linking pairs of E3BP molecules across the surface of the E2 core assembly. Low resolution solution structures obtained for rE2/E3BP, bE2/E3BP and tLi19/tLi30 by small angle x-ray scattering (SAXS) and SANS revealed the presence of icosahedral cores with open pentagonal faces favouring the substitution model of core organisation. These solution structures also indicated high structural similarity between the recombinant and native cores, as well as with the crystal structure obtained previously for the truncated bacterial E2 core. In addition, homology modelling and superimpositions of high- and low-resolution structures of the core revealed conservation of the overall pentagonal dodecahedral morphology despite evolutionary diversity. Evidence for the substitution model of core organisation was further substantiated by negative stain EM of the recombinant and bovine E2/E3BP cores. SANS stoichiometry data indicated the binding of 10 E3 dimers per E2/E3BP core. Although this could correspond to approximately 1:1 stoichiometry between E2/E3BP:E3, subsequent radiolabelling studies suggested possible variation in core subunit composition between the native and recombinant E2/E3BP cores. Therefore, as opposed to the 48E2+12E3BP substitution model based on AUC and SAXS studies with the recombinant E2/E3BP core, rE2/E3BP cores produced in this study indicated a higher level of incorporation of E3BPs with a maximum core composition of 40E2+20E3BP. On the basis of this new finding we have proposed the ‘variable E3BP substitution model’, wherein the number of E3BPs within the core can range from 0 to a maximum of 20, thus resulting in variable populations of E2/E3BP cores. Despite this core variability, the highly controlled regulatory mechanisms in vivo may bias the core composition towards an average of 48E2+12E3BP. However, as the over-expression of the recombinant E2/E3BP core in our study is not as tightly regulated as in vivo, higher number of E3BPs (>12) is observed to be integrated into the core. This new level of architectural complexity and variable subunit composition in mammalian PDC core organisation is likely to have important implications for the catalytic mechanism, overall complex efficiency and tissue-specific regulation by the intrinsic PDC kinases (PDKs) in normal and disease states. The E2 cores of the PDC family are known to be highly flexible, exhibiting inherent size variability reflective of the ‘breathing’ of the core. Integration of E3BP into the E2 core assembly would then be expected to have significant consequences for the structural assembly, affecting the ‘breathing’ and in turn the function and regulation of the complex. Unfolding studies to assess core stability via circular dichroism (CD) and tryptophan fluorescence revealed lower stability of the rE2/E3BP core as compared to cores composed exclusively of rE2 subunits, thus implying the contribution of E3BP towards core destabilisation. In addition, crosslinking studies indicated weak dimerisation of rE3BP, which may be a key factor promoting core destabilisation. The lower stability of the E2/E3BP core may be of benefit in mammals where sophisticated fine tuning is required to obtain cores with optimal catalytic and regulatory efficiencies. SAXS solution structures of E2/E3BP cores obtained were unable to locate the exact positions of E3BP within the core. However, SANS in combination with contrast matching of selectively deuterated components as well as cryo-EM, EM tomography and single molecule studies could be used in future for determination of the exact locations of E3BP, and validating the importance of E2/E3BP core organisation and subunit composition for overall PDC function and regulation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Glycolysis, SANS, SAXS, AUC, Pyruvate Dehydrogenase Complex (PDC)
Subjects: Q Science > QC Physics
Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Byron, Dr. Olwyn and Lindsay, Prof. Gordon
Date of Award: 2009
Depositing User: Ms Swetha Vijayakrishnan
Unique ID: glathesis:2009-573
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 Mar 2009
Last Modified: 10 Dec 2012 13:19
URI: http://theses.gla.ac.uk/id/eprint/573

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