Stoichiometry of binding of mature and truncated forms of the dihydrolipoamide dehydrogenase-binding protein to the dihydrolipoamide acetyltransferase core of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

The dihydrolipoamide dehydrogenase-binding protein (E3BP), a component of the Saccharomyces cerevisiae and mammalian pyruvate dehydrogenase (PDH) complexes, anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2). To gain further insight into the number and localization of binding sites for E3BP on the 60-mer E2, truncated forms of the E3BP lacking the lipoyl and E3-binding domains were engineered by deletion mutagenesis. The recombinant proteins contained a polyhistidine extension on the amino terminus to facilitate purification to near-homogeneity. The stoichiometry of binding of the truncation mutants to a truncated form (inner core) of E2 (tE2, residues 181-454), lacking the lipoyl domain and the E1-binding domain, was determined. Mixtures containing tE2 and excess intact or truncated forms of E3BP were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or tE3BP, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that tE2 binds about 20 copies of intact E3BP-H, about 24 copies of tE3BP-H144 (residues 144-380), lacking the lipoyl domain, and about 31 copies of tE3BP-H218 (residues 218-380), lacking both the lipoyl and E3-binding domains. The results indicate that there apparently is a binding site for E3BP on each E2 subunit and that steric hindrance by segments of E3BP prevents full stoichiometric binding of E3BP to the pentagonal dodecahedron-like E2.