3D electron microscopy deposition and protein reveals the variable dynamics of the peripheral pyruvate dehydrogenase component about the core

Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E-1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC). Structures representative of PDC preparations with approximate to40% and full E-1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes. The reconstructions derived from two size groups showed that the deposition of the E-1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E-1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC. This study also shows that the linker of dihydrolipamide acetyltransferase (E-2) that tethers E-1 to the E-2 core increases in length from approximate to50 to 75 Angstrom, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E-1 occupancy of its 60 E-2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E-1 molecules. However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E-1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E-1. The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.