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

Abstract

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 Ϸ40% 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 E1 to the E2 core increases in length from Ϸ50 to 75 Å, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E 1 occupancy of its 60 E2 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 E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1. 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.A central feature of eukaryotic pyruvate dehydrogenase complexes (PDCs) is a 60-mer core with the morphology of a pentagonal dodecahedron (1-4). The dihydrolipoamide acetyltransferase (E 2 ) component serves as a scaffold to which the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ) components are attached. The E 2 dodecahedron consists of sets of three tightly bound subunits at each of its 20 vertices. The trimers are interconnected by 30 tenuous and flexible bridges that enable the core to breathe as evidenced by its size variability of Ϸ17% (240-280 Å in diameter) at room temperature (5).3D reconstructions of subcomplexes of the Saccharomyces cerevisiae E 2 core have revealed that 12 E 3 components are attached by a binding protein (BP) inside the 12 pentagonal openings of the cage-like E 2 (6), whereas the E 1 components form a shell that surrounds the underlying E 2 core (7-9). The reconstructions show that the 60 E 2 subunits are organized in 20 trimers. Each E 2 subunit contains an Ϸ50-Å-long linker that anchors an E 1 tetramer. Three of these linkers emanate from the outside edges of the triangular-shaped base of the E 2 trimer and form a cage around its base that may shelter the lipoyl domains of E 2 and the E 1 and E 2 active sites. We proposed that the lipoyl domain and its tether (swinging arm) rotates about the E 1 -binding domain of E 2 , which...

Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy

“…This unexpectedly organized architecture could promote highly efficient active site coupling by confining the trajectories of the lipoyl domains as they shuttle between the successive active sites. A somewhat similar architecture has been reported for the PDH complexes from bovine kidney (10) and Saccharomyces cerevisiae (11). Here, we extend our previous studies with the report of the architecture of a 1:1 stoichiometric complex containing 60 copies each of the E2 and E3 enzymes.…”

“…Thus cryoelectron microscopy of the PDH complex from bovine kidney (10) and S. cerevisiae (11) indicates that the E1 components form a radial shell ϳ50 Å above the central E2 core, although it is reported in the former paper that a high occupancy of the E1 binding sites can lead to an extension of the linker region from 50 to ϳ75 Å. Moreover, in these complexes, the E3 homodimers are bound to the E2 core by association with an additional component, an E3-binding were used.…”

Strep-tag II and Twin-Strep Based Cassettes for Protein Tagging by Homologous Recombination and Characterization of Endogenous Macromolecular Assemblies in Saccharomyces cerevisiae

“…This core complex is surrounded by the PDH E1 subunit (consisting of Pda1 or E1A and Pdb1 or E1B), which forms a coat-like structure [48,49]. This E1 coat consists of multiple E1 subunits arranged around an E2 60-mer [48]. The assembled complex has been reported to sediment at about 70-77S, and thus is one of the largest enzymes found in eukaryotic cells [46,50].…”

“…The E2 complex is connected to 12 E3 dihydrolipoamide dehydrogenase subunits (Lpd1) via the E3 binding protein (Pdx1) [47]. This core complex is surrounded by the PDH E1 subunit (consisting of Pda1 or E1A and Pdb1 or E1B), which forms a coat-like structure [48,49]. This E1 coat consists of multiple E1 subunits arranged around an E2 60-mer [48].…”

Other conceivable renditions of some of the oxidative processes used in the biosynthesis of steroid hormones

“…Reactions (1)-(5) have all been experimentally documented. Reaction (6), the formation of a ␣-ketol from a bisalkoxy radical is hypothetical but can be deduced from reaction (5). 1 For more on multienzyme complexes [5,6].…”