Using cryo-electron microscopy, we have extended our previous determination of the structure of the E1E2 complex with a detailed analysis of the E2 core decorated with 60 copies of the homodimeric 100 kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of about 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain. In a related set of studies we have addressed another important unresolved question, which is to determine whether the gap between the central E2 core and the outer enzyme shell is maintained by virtue of protein-protein interactions in the outer shell or by stiffness of the linker region connecting the core to the peripheral subunit binding domain. Using a combination of circular dichroism, analytical ultracentrifugation and solution NMR studies we have obtained evidence that the peptide corresponding to the linker region has an extended conformation with a persistence length of 75-89 , consistent with the observed size of the gap. Cryo electron tomography of individual complexes with varying occupancies of enzymes in the outer shell confirmed unequivocally that the annular between the core and the outer shell was maintained even at very low E1 or E3 occupancies. These studies demonstrate unambiguously that it is the linker, rather than interactions between the outer shell enzymes, that are responsible for holding the subunits above the core. We conclude that the inner linker region of PDH enzymes are critical structural elements, serving to maintain the annular gap required for coupling the decarboxylation of pyruvate in the outer shell to the synthesis of acetyl CoA in the core. Our work thus reveals unique insights into the architecture and inner workings of a fascinating cellular machine that is a paradigm among multi-enzyme complexes that function using mobile swinging arms to couple distantly separated active sites. More broadly, our demonstration of mapping individual enzymes within single multi-enzyme complexes will likely prove to be an invaluable approach to obtain structural information, without molecular averaging, on large and structurally heterogeneous biological assemblies that are not amenable to analysis by NMR or X-ray crystallographic techniques.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010412-09
Application #
7733026
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2008
Total Cost
$248,857
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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