On the Unique Structural Organization of the Saccharomyces cerevisiae Pyruvate Dehydrogenase Complex

Abstract

Dihydrolipoamide acyltransferase (E 2 ), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of ␣-keto acids, forms the central core to which the other components attach. We have determined the structures of the truncated 60-mer core dihydrolipoamide acetyltransferase (tE 2 ) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex and complexes of the tE 2 core associated with a truncated binding protein (tBP), intact binding protein (BP), and the BP associated with its dihydrolipoamide dehydrogenase (BP⅐E 3 ). The tE 2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges. Previous studies have given rise to the generally accepted belief that the other components are bound on the outside of the E 2 scaffold. However, this investigation shows that the 12 large openings in the tE 2 core permit the entrance of tBP, BP, and BP⅐E 3 into a large central cavity where the BP component apparently binds near the tip of the tE 2 trimer. The bone-shaped E 3 molecule is anchored inside the central cavity through its interaction with BP. One end of E 3 has its catalytic site within the surface of the scaffold for interaction with other external catalytic domains. Though tE 2 has 60 potential binding sites, it binds only about 30 copies of tBP, 15 of BP, and 12 of BP⅐E 3 . Thus, E 2 is unusual in that the stoichiometry and arrangement of the tBP, BP, and E 3 ⅐BP components are determined by the geometric constraints of the underlying scaffold.Pyruvate dehydrogenase complexes (PDCs) 1 are among the largest (M r ϳ10 6 -10 7 ) and most complex multienzyme structures known. They consist of a central core that has both functional and structural roles in organizing the complex, the dihydrolipoamide acetyltransferase (E 2 ) subunits associate to form the core complex that also serves as a scaffold to which the other components are attached (1-4). Electron microscopy (5-8) and x-ray crystallography (9 -11) studies have revealed two fundamental morphologies of the E 2 cores. The cubic E 2 core of the Escherichia coli PDC has 24 subunits arranged with octahedral symmetry, whereas the pentagonal dodecahedral E 2 core of the PDC complexes from eukaryotes and some Grampositive bacteria has 60 subunits arranged with icosahedral symmetry. The subunits form cone-shaped trimers at each of the 8 and 20 vertices of the cubic and dodecahedral structures, respectively. These trimers are interconnected by bridges to form a cage-like complex (8 -11).The E 2 subunit is a multidomain structure to which the other constituents of the functional PDC (1-4) bind (see Fig. 1). These include the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ). E 3 requires a binding protein (BP) to anchor it to the core of the yeast (12) and mammalian PDCs (13-15) though, in E. coli and Bacillus stearothermophilus PDCs, BP is not required (1-4).It is widely held that the constituent proteins are bound to the out...

The Dihydrolipoamide S-Acetyltransferase Subunit of the Mitochondrial Pyruvate Dehydrogenase Complex from Maize Contains a Single Lipoyl Domain

“…Formation of this core is further supported by electron microscopy showing icosahedral symmetry of the maize E2. This report is the first example of a plant E2 core visualized by electron microscopy and establishes that the icosahedral symmetry of the plant mitochondrial E2 core is most likely identical to that for animal (14), fungal (15), and Gram-positive bacterial (12,13) PDCs. Furthermore, the size of the maize E2 cores visualized by electron microscopy were about the same as those from other organisms.…”

“…None of the protein complexes were cubic in shape, and most of them appeared "six-sided." The E2 complexes from animals and yeast, which are both pentagonal dodecahedrons, gave this same appearance (14,15). Upon closer inspection, the three axes of symmetry characteristic of a pentagonal dodecahedron were observed (Fig.…”

“…The E2 subunit forms the core to which all other PDC subunits bind (10). In Gram-negative bacteria such as Escherichia coli, the oligomeric core contains eight E2 trimers arranged as a cube with octahedral symmetry (11), while in Gram-positive bacteria (12,13) and eukaryotes (14,15) the core contains 20 E2 trimers arranged as a pentagonal dodecahedron with icosahedral symmetry. Amino acid sequence alignments comparing E2s from divergent organisms revealed that they are multi-domain proteins (see Ref.…”

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

“…This appears to lead to the E3 being more intimately associated with the inner E2 core in the openings on the 12 5-fold faces of the dodecahedron (8,10). Other cryoelectron microscopic (9) and biochemical studies (40) indicate a more exterior positioning of the E3 in the mammalian PDH complex.…”

Apparent mitochondrial asymmetry in Xenopus eggs

“…5A) contains two lipoic acid binding domains that are highly homologous to those found in PDC-E2 from other vertebrates (Thekkumkara et al, 1988;Matuda et al, 1992). In addition to its central catalytic role, PDC-E2 is also the structural core of the complex (Stoops et al, 1997) and contains domains that mediate interactions with PDC-E1 and PDC-E3 (Fig. 5A).…”