Organization of the Cores of the Mammalian Pyruvate Dehydrogenase Complex Formed byE2 andE2 Plus theE3-binding Protein and Their Capacities to Bind theE1 andE3 Components
Journal of Biological Chemistry
The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the Cterminal 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
... ase (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 E2⅐E3BP was thought to be 60 E2 plus ϳ12 E3BP. We have prepared homogenous human components. E2 and E2⅐E3BP have s 20,w values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2⅐E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2⅐E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound ϳ60 E1 and maximally sedimented 64.4 ؎ 1.5 S faster than E2, whereas E1-saturated E2⅐E3BP maximally sedimented 49.5 ؎ 1.4 S faster than E2⅐E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (ϳ12) were bound by E2⅐E3BP than by E2. The findings of a smaller E2⅐E3BP 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 E2 48 ⅐E3BP 12 mass, which is consistent with this model. . 1 The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltrans-ferase component; L1 domain, NH 2 -lipoyl domain of E2; L2 domain, interior lipoyl domain of E2; B, binding domain in E2 for E1; H1, H2, and H3 are the linker (hinge) regions of E2 (Fig. 1A); scE2, E2 with PreScission protease site introduced in H3 domain of E2; t-E2, truncated form of E2 with I domain and small part of H3 linker region; E3, dihydrolipoyl dehydrogenase; E3BP, E3-binding protein; L3, lipoyl domain of E3BP; BЈ, binding domain of E3BP that binds E3; IЈ domain, inner domain of E3BP; H1Ј and H2Ј linker (hinge) regions in E3BP (Fig. 1A) ; AUC, analytical ultracentrifugation; SAXS, small angle x-ray scattering; R 0 , R s , R g , and R e are, respectively, the unhydrated spherical Stokes radius, Stokes radius, radius of gyration, and particle excluded volume radius.