Why are the 2-oxoacid dehydrogenase complexes so large? Generation of an active trimeric complex.

The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures.

The catalytic core of an archaeal 2-oxoacid dehydrogenase multienzyme complex is a 42-mer protein assembly.

The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 Å. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications.