title: Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components.
abstract: The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I' domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2.E3BP was thought to be 60 E2 plus approximately 12 E3BP. We have prepared homogenous human components. E2 and E2.E3BP have s(20,w) values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2.E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2.E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound approximately 60 E1 and maximally sedimented 64.4 +/- 1.5 S faster than E2, whereas E1-saturated E2.E3BP maximally sedimented 49.5 +/- 1.4 S faster than E2.E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (approximately 12) were bound by E2.E3BP than by E2. The findings of a smaller E2.E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I' domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248.E3BP12 mass, which is consistent with this model.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Hiromasa
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Fujisawa
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Aso
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Roche
mesh_terms: D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry*; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000737:chemistry*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry*; D012542:Scattering, Radiation; D014461:Ultracentrifugation; D014965:X-Rays
chemical_list: C045867:PDHX protein, human; D010455:Peptides; D011768:Pyruvate Dehydrogenase Complex; D030481:Pyruvate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase; D051047:Dihydrolipoyllysine-Residue Acetyltransferase