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The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (M r Ϸ 7.8 ؋ 10 6 ) comprising about 22 molecules of pyruvate dehydrogenase (E 1) and about 6 molecules of dihydrolipoamide dehydrogenase (E 3) with its binding protein associated with the 60-subunit dihydrolipoamide acetyltransferase (E2) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetylCoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E 1 molecules bound to one E2 trimer adjacent to an E3 molecule in each of 12 pentagonal openings. The E1 components are anchored to the E 1-binding domain of the E2 subunits through an Ϸ50-Å-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E 2 trimer and form a cage around its base that may shelter the lipoyl domains and the E 1 and E2 active sites. The docking of the atomic structures of E1 and the E 1 binding and lipoyl domains of E2 in the electron microscopy map gives a good fit and indicates that the E 1 active site is Ϸ95 Å above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E 1-binding domain of E 2, which is centrally located 45-50 Å from the E1, E2, and E3 active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites. T he pyruvate dehydrogenase complex (PDC) serves as the link between glycolysis and the tricarboxylic acid cycle and generally has prominence in the description of these metabolic pathways because they serve as a major source of cellular energy. A central feature of PDCs is a 24-mer (Escherichia coli) or 60-mer (eukaryotes and some Gram-positive bacteria) core with the morphologies of a cube or pentagonal dodecahedron, respectively (1-4). The structures with the latter morphology comprise the largest (M r Ϸ 10 7 ) multienzyme complexes known. Even more remarkable than their exceptional size and morphology, these complexes encompass some of the most unusual features found in structural biology as described below.The E 2 core comprises the dihydrolipoamide acetyltransferase activity and is the only oligomeric enzyme complex known to be organized with the shape of a pentagonal dodecahedron. Moreover, the 250-Å-diameter dodecahedron has a very unusual feature: the tightly bound trimers at each of its 20 vertices seem to be interconnected by 30 flexible bridges enabling the core to ''breathe,'' as evidenced by an extraordinary size variability of 40 Å (17%) at room temperature. The breathing core apparently is a common feature in the phylogeny of the PDCs, suggesting that protein dynamics is an integral component of the function of these multienzyme complexes (5). Moreover, dodecahedral morphology of the core favors a synchronous or harmonious change in the length of the bridges that is relate...

Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE 2 ) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE 2 forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE 2 in the same field vary in size ϳ20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-Å difference in the diameter of the smallest and largest structures that corresponds to ϳ14 Å of variation in the length of the bridge between interconnected trimers. Companion studies of mature E 2 show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE 2 shows that there is an ϳ10-Å gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.The pyruvate dehydrogenase complexes (PDCs) 1 are among the largest (M r ϳ10 6 to 10 7 ) and most complex multienzyme structures known. A central feature of these complexes is a 24-mer (Escherichia coli) or 60-mer (eukaryotes and some Gram-positive bacteria) dihydrolipoamide acetyltransferase (E 2 ) core with the morphologies of a cube or a pentagonal dodecahedron, respectively (1-4). The cores have both functional and structural roles in organizing the multienzyme complex; the E 2 activity is associated with the scaffold to which the other components are attached. These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ), which requires a binding protein (BP) to anchor it to the core of the yeast and mammalian PDCs, although, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1-4).The E 2 subunits have multidomain structures consisting of one, two, or three amino-terminal lipoyl domains, followed by an E 1 and/or E 3 binding domain, and a carboxyl-terminal catalytic domain (1-4). X-ray crystallography (5-9) and threedimensional electron microscopy (10, 11) show that the E 2 catalytic domains are arranged in cone-shaped trimers at each of the 8 or 20 vertices of the cubic or dodecahedral structures, respectively (7,8,10,11). The trimers are interconnected by bridges to form an empty cage-like complex with the tip of the trimer directed toward the center of the structure.Examination of the 4-Å resolution crystal structures of dodecahedral truncated E 2 (tE 2 ) cores from Enterococcus faecalis and B. stearothermophilus and the 2-Å resolution crystal structure of a cubic tE 2 core from Azotobacter vinelandi...

Although the pathogenesis of Alzheimer's disease (AD) is not fully understood, growing evidence indicates that the deposition of beta-amyloid (Abeta) and the local reactions of various cell types to this protein play major roles in the development of the disease. Immunization with the Abeta 1-42 peptide has been reported to decrease Abeta deposits in the brains of mutant amyloid precursor protein (APP/V717F) transgenic (tg) mice (Schenk et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999;400:173-177). We have replicated this finding in APPswe/PS1DeltaE9 tg mice, which also develop Abeta deposits in the brain. The immunized animals developed high titers of antibodies against Abeta 1-42 in serum, and Abeta deposits in the brains were significantly reduced. Using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry and ProteinChip((R)) technology, we detected trends toward increased soluble Abeta peptide in the brain and a decrease in assayable Abeta peptide in the serum of immunized compared with control animals. This last finding raises the possibility that anti-Abeta antibodies in the periphery sequester Abeta peptides or target them for degradation and in this way contribute to the enhanced Abeta clearance from the brain in immunized animals.

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...