Molecular Architecture of Pyruvate Dehydrogenase Complexes

Abstract

Pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional protein assemblies within cells, catalyzing the four-step conversion of pyruvate to acetyl CoA using an assembly comprised of multiple copies of E1, E2 and E3 enzymes, Using cryo-electron microscopy, we have shown that the outer shell enzymes (E1 decarboxylase and E3 pyruvate dehydrogenase) are separated from the inner icosahedral core (E2 acetyl transferase) by an annular gap that is about ∼ 100 A wide. The presence of the gap is a key structural element in the spatial organization of the three enzymes in the complex, and allows the “swinging arm” lipoyl domain to shuttle between the active site of the E2 at the inner core and the active sites of E1 and E3 enzymes at the outer shell to carry out synthesis of acetyl CoA. Using a combination of circular dichroism, analytical ultracentrifugation and solution NMR studies we have also demonstrated that the peptide corresponding to the linker region has an extended conformation with a persistence length of ∼75-89 Angstroms, 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 gap 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. The prospect of using cryo-electron tomography to map the locations of individual enzymes within single multi-enzyme complexes could be a powerful 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.