Stoichiometry of Binding of Mature and Truncated Forms of the Dihydrolipoamide Dehydrogenase-Binding Protein to the Dihydrolipoamide Acetyltransferase Core of the Pyruvate Dehydrogenase Complex from <i>Saccharomyces cerevisiae</i>

Self Cite

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...

Section: Methodsmentioning

confidence: 99%

Direct Evidence for the Size and Conformational Variability of the Pyruvate Dehydrogenase Complex Revealed by Three-dimensional Electron Microscopy

Zhou¹,

Liao²,

Cheng

et al. 2001

Self Cite

“…Protein Preparations-The S. cerevisiae genes or subgenes encoding tE 2 , tBP, BP, and E 3 were expressed in E. coli, and the recombinant proteins were purified to homogeneity as described (12,18). The tE 2 subunit (residues 206 -454) had Ser-Gly at the N terminus.…”

Section: Methodsmentioning

confidence: 99%

“…6). The more highly condensed distribution of BP in the cavity could explain the ϳ2-fold decrease in its binding to tE 2 compared with tBP (12,18). Apparently, more copies of the extended tBP structure may access binding sites inside the core.…”

Section: Tbp Bp and Bp⅐e3 Morphologies And Interactions With Thementioning

confidence: 99%

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

Stoops

Cheng

Yazdi

et al. 1997

Self Cite

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 putative catalytic site histidine residue present in the inner core domains of all dihydrolipoamide acyltransferases is replaced by a serine residue in human E 3 BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely. Coexpression of cDNAs for E 3 BP and E 2 resulted in the formation of an E 2 ⅐E 3 BP subcomplex that spontaneously reconstituted the pyruvate dehydrogenase complex in the presence of native E 3 and recombinant pyruvate decarboxylase (E 1 ).…”

mentioning

confidence: 99%

Dihydrolipoamide Dehydrogenase-binding Protein of the Human Pyruvate Dehydrogenase Complex

Harris

Bowker-Kinley

et al. 1997

100

Protein X, recently renamed dihydrolipoamide dehydrogenase-binding protein (E 3 BP), is required for anchoring dihydrolipoamide dehydrogenase (E 3 ) to the dihydrolipoamide transacetylase (E 2 ) core of the pyruvate dehydrogenase complexes of eukaryotes. DNA and deduced protein sequences for E 3 BP of the human pyruvate dehydrogenase complex are reported here. With the exception of only a single lipoyl domain, the protein has a segmented multi-domain structure analogous to that of the E 2 component of the complex. The protein has 46% amino acid sequence identity in its amino-terminal region with the second lipoyl domain of E 2 , 38% identity in its central region with the putative peripheral subunit-binding domain of E 2 , and 50% identity in its carboxyl-terminal region with the catalytic inner core domain of E 2 . The similarity in the latter domain stands in contrast to E 3 BP of Saccharomyces cerevisiae, which is quite different from its homologous transacetylase in this region. The putative catalytic site histidine residue present in the inner core domains of all dihydrolipoamide acyltransferases is replaced by a serine residue in human E 3 BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely. Coexpression of cDNAs for E 3 BP and E 2 resulted in the formation of an E 2 ⅐E 3 BP subcomplex that spontaneously reconstituted the pyruvate dehydrogenase complex in the presence of native E 3 and recombinant pyruvate decarboxylase (E 1 ).The pyruvate dehydrogenase complex (PDC) 1 catalyzes the oxidative decarboxylation of pyruvate with the formation of CO 2 , acetyl-CoA, and NADH. The eukaryotic complex has 30 copies of a tetrameric (␣ 2 ␤ 2 ) pyruvate decarboxylase (E 1 ) component noncovalently bound along the edges of an icosahedral 60 meric dihydrolipoamide acetyltransferase (E 2 ) core (1). Twelve copies of a homodimeric dihydrolipoamide dehydrogenase (E 3 ) component are believed held on the faces of the E 2 core by a corresponding number of monomeric E 3 -binding proteins (protein X, E 3 BP) (2, 3). With the exception of E 3 BP, the role of each enzymatic component in the overall reaction catalyzed by the complex is basically understood. E 1 catalyzes a thiamine diphosphate-dependent oxidative decarboxylation of pyruvate and the reductive acetylation of a lipoyl residue covalently attached to the lipoyl domain of E 2 . E 2 then catalyzes transfer of the acetyl group to coenzyme A, leaving a reduced E 2 lipoyl group that the E 3 component uses as an electron source for FAD-dependent reduction of NAD ϩ to NADH. Called protein X because its function was not apparent at the time it was originally discovered to be a component of eukaryotic PDCs (4 -6), E 3 BP has generated considerable interest because of sequence similarity with E 2 (7-10) and evidence that it has a covalently bound lipoyl moiety (4,6,8,11). Its function was difficult to establish in early studies because of a very tight association with the E 2 core of the complex. Very elegant limited proteolysis and immunological studies...