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

Yasuaki Hiromasa, Tetsuro Fujisawa, Yoichi Aso, Thomas E. Roche
2003 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
more » ... 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.
doi:10.1074/jbc.m308172200 pmid:14638692 fatcat:x2j4kglsorelbi2ufjqznfy5km