Molecular architecture of the human pyruvate dehydrogenase complex.

Mammalian pyruvate dehydrogenase multi-enzyme complex (PDC) is a key metabolic assembly responsible for the maintenance of glucose homeostasis. PDC comprises a central pentagonal dodecahedral core of 60 dihydrolipoamide acetyltransferase (E2) and 12 E3 binding protein (E3BP) molecules. E2 is thought to form the edges of the icosahedral core structure while a single E3BP molecule is thought to occupy each of the 12 pentagonal faces. Both E2 and E3BP have a modular multi-domain organisation and are responsible for the attachment of up to 30 pyruvate decarboxylase (El) heterotetramers and 6-12 dihydrolipoamide dehydrogenase (E3) homodimers, respectively. The formation of highly specific E2/E1 and E3BP/E3 subcomplexes is characteristic of PDC from eukaryotes and critical for normal complex function. This thesis describes the large-scale purification of tagged, recombinant human PDC proteins E2, E3 and E3BP as well as several truncated E2 and E3BP constructs. Due to the limited solubility of recombinant human El, the protein was obtained by purification from native bovine PDC. The ability to purify relatively large amounts of proteins enabled the characterisation of the individual proteins as well as their subcomplexes using a variety of biochemical and biophysical techniques. Truncated E3BP, full-length E3 and their resultant subcomplex were analysed in solution by analytical ultracentrifugation (AUC). The stoichiometry of interaction was determined to be 2:1 (E3BP:E3) using native polyacrylamide gel electrophoresis (PAGE), AUC, isothermal titration calorimetry (ITC) and small angle x-ray scattering (SAXS), thus implying the existence of a network of E3 "cross-bridges" linking pau's of E3BP molecules across the surface of the E2 core assembly. A low resolution structure for E3 obtained by SAXS shows significant differences to the crystal structures obtained previously for human and yeast E3s. The low resolution structure determined for the truncated E3BP/E3 subcomplex was surprisingly anisometric, indicating the asymmetric distribution of lipoyl domains within the subcomplex. Rigid-body modelling with homology models of E3 and the individual E3BP domains resulted in a structure in which only one of the E3BP lipoyl domains was docked into the E3 active site. This new level of architectural complexity in mammalian PDC has important implications for the catalytic mechanism, overall complex efficiency and regulation by its intrinsic PDC kinase. Analogous to the E3BP/E3 subcomplex, initial investigations of the interaction of truncated E2 with El using AUC also seem to indicate the formation of 2:1 subcomplexes, cross-linking neighbouring E2 molecules on the PDC core surface. However, the relatively low stability of bovine El - as revealed by AUC - has hampered the investigation of E2/E1 subcomplex formation. Until recently, mammalian PDC core was thought to consist of 60 E2 and 12 E3BP molecules (the "addition" model). The relatively recent publication of an alternative, "substitution" model by Hiromasa and colleagues (2004), where E3BP replaces 12 E2 molecules, resulting in a 48:12 (E2:E3BP) core structure, initiated the search for experimental approaches able to distinguish between the PDC core models. SAXS data, even in conjunction with ab initio reconstruction techniques were deemed insufficient for this purpose, while small angle neutron scattering (SANS) in combination with contrast matching of selectively deuterated components gave promising initial results and could be used in future for the resolution of this fundamental problem in PDC subunit organisation.