The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes

Abstract

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (M(r) approximately 7.8 x 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 (E(2)) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetyl-CoA 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 E(2) trimer adjacent to an E(3) molecule in each of 12 pentagonal openings. The E(1) components are anchored to the E(1)-binding domain of the E(2) subunits through an approximately 50-A-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 E(2) active sites. The docking of the atomic structures of E(1) and the E(1) binding and lipoyl domains of E(2) in the electron microscopy map gives a good fit and indicates that the E(1) active site is approximately 95 A 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 A from the E(1), E(2), and E(3) active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites.

Figure 1

CryoEM images. Fields of images of frozen-hydrated bovine PDC (A) and S. cerevisiae tE₂ (residues 221–454; B) and E₂ (C). The E₁ molecules appear equally distributed about the scaffold, and their association with its outside increases the diameter from ≈250 to ≈500 Å. The similarity between the tE₂ and E₂ images indicates that N-terminal domains (residues 1 to ≈220) are flexible.

Figure 2

3D reconstruction of bovine PDC (A and B) and diagrammatic representation of the structural domains of E₂ subunit (C). Shaded-surface representation of 3-fold axes of symmetry of the 3D reconstruction of the bovine kidney PDC (A) and with the closest half removed to reveal the linker (blue) that binds E₁ (yellow) to the E₂ core (green; B). The inner linker is ≈50 Å in length. (C) The C-terminal self-association domain is responsible for the assembly of the dodecahedral scaffold to which E₁ and BP⋅E₃ bind. The N-terminal half of the E₂ comprises the L₁ and L₂ lipoyl domains, and the E₁-binding domain, and their associated linkers. The inner linker is revealed in the 3D structure of the PDC.

Figure 3

Shaded-surface representations of 3-fold view of E₂ core. (A) E₁ was removed from PDC to reveal the underlying E₂ core (green) and its inner linkers (light blue). An E₂ trimer is outlined by the yellow square, and the 5-, 3-, and 2-fold axes are indicated. The linkers change the shape of the trimer base so that it appears that they are connected to its corners. (B) Superposition of the cut-away cryoEM PDC structure and the x-ray structure of B. stearothermophilus tE₂. The atomic model of the tE2 dodecahedral core was directly superimposed on the core of the cryoEM structure by aligning the icosahedral 5-, 3-, and 2-fold axes with those of the cryoEM structure. For clarity, only one of the 20 tE₂ trimers of the atomic model is shown. The dark and light blue and green x-ray ribbon structure represent the three identical subunits that comprise the tE₂ core. The three N-terminal linkers are directly opposite the N-terminal helix (H1) near the middle of the outside edge of the trimer base as indicated, and the N-terminal loop is directed toward the 3-fold axis. The bridge interconnects adjacent trimers to form the dodecahedral core.

Figure 4

Cut-away representation of the structural unit of bovine kidney PDC. (A) Top view of the cut-away structure comprising E₂ trimer and its associated three E₁ tetramers. (B) Side view of A. The three E₁ tetramers, E₂ trimer, and its linkers form a cage that encloses the E₁ and E₂ active sites. (C) Top view of wire-frame representation of the structure in A in which the atomic structures of P. putida E₁ (light blue) and the B. stereothermophilus lipoyl domains (orange, denoted by arrows in D) and the putative E₁-binding domain (bold red) are docked in the EM envelope of bovine E₂. The E₁ tetramer was docked in the cryoEM envelope so that the E₁-binding domain of E₂ and its inner linker is close to the 2-fold axis of E₁ where the C-terminal domains of the β subunits meet (9). We propose that the 60 potential centers for acetyl-CoA synthesis are organized in sets of three comprising three E₂ subunits and three E₁ tetramers. (D) Side view of C in which the B. stearothermophilus tE₂ x-ray structure is superimposed as described in Fig. 3. The docked ribbon structure of P. putida E₁ (light blue) shows the spatial relationship of its crystallographic 2-fold axis to the 3-fold axis of B. stearothermophilus tE₂. The E₁ active sites are about 95 Å above the E₂ catalytic site on the trimer base.

Figure 5

Cut-away model of the fully assembled PDC viewed on its 3-fold axis. The E₃ homodimer (red) of A. vinelandii x-ray structure (32) filtered to 20Å resolution was docked into the pentagonal opening of the core (green) according to studies of the S. cerevisiae PDC (7). The BP⋅E₃ components associated with the core are not revealed in the shaded-surface representation of the bovine kidney PDC at this threshold because only about 6 molecules of E₃ are bound. A radial density plot analysis of the complex shows a peak of density inside the core which corresponds to the position of BP⋅E₃ peak in S. cerevisiae tE₂ (ref. ; data not shown). The inner linkers (light blue) bind E₁ (yellow) to the E₂ scaffold (green). The E₁-binding site on the E₂ inner linker is located ≈50 Å above the scaffold as indicated by “*” and serves as the anchor for the lipoyl domains to pivot. The structure shows that the swinging arm pivots about a position that is ≈50 Å from the E₁, E₂, and E₃ active sites.