Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion

Abstract

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.

Fig. 1. Reaction mechanism and cryo-EM reconstruction of E. coli PDHc.

a Scheme of the reactions catalyzed by the individual components of PDHc. b Overall reaction catalyzed by PDHc. c High-resolution cryo-EM reconstruction of the inner E2p core in alignment with the low-resolution reconstruction of the outer shell of the complex. Catalytic cores of E2p are colored magenta, lipoyl domains cyan, and the outer shell components (E1p/E3) yellow. d Ball-and-stick representation of the dihydrolipoyllysine moiety. e Scheme of the domain organization of E2p. f A simplified scheme of the covalent swinging-arm substrate channeling mechanism in the catalytic cycle of PDHc. Only one LD is shown and only one copy of each enzyme is shown, but the LDs can reach multiple copies of each enzyme in the multienzyme complex. PSBD can bind to either E1p or E3. E1p—pyruvate dehydrogenase, E2p—dihydrolipoyl transacetylase, E3—dihydrolipoyl dehydrogenase, LD—lipoyl domain, PSBD—peripheral subunit-binding domain. Panels d–f were prepared based on Arjunan et al..

Fig. 2. Structure of the 24-mer of E. coli dihydrolipoyl transacetylase (E2p).

a Surface representation of the overall structure of the E2p cubic core with each E2p trimer shown in a different color; E2p monomers in the top left E2p trimer are shown in different shades of cyan. b Cartoon structural representation of the E2p trimer. In the top E2p monomer the catalytic domain is colored magenta and the associated lipoyl domain in cyan with the dihydrolipoyllysine residue depicted as spheres, while in the other two E2p monomers the catalytic and lipoyl domains are colored in different shades of green and blue, respectively. c Molecular details of the contact interface between E2p trimers viewed along the two-fold axis. Interface residues located within the van der Waals distance are shown as sticks with residue numbers indicated for one E2p monomer (cyan) in black and for the second monomer (magenta) in gray. Pale cyan dashed lines represent hydrogen bonds and yellow dashed lines represent other polar interactions.

Fig. 3. Interaction of the lipoyl domain with the catalytic domain of E. coli dihydrolipoyl transacetylase.

The color code for the individual domains corresponds to Fig. 2b. a Acidic patch on the surface of lipoyl domain interacts with the positive dipole and an arginine residue in the N-terminal tip of the catalytic domain helix H1, and with two basic residues from the N-terminal stretch of the neighboring catalytic domain. Residue D213 lies on the backside of the lipoyl domain. b Key interactions are visualized between the loops of the lipoyl and catalytic domains at the binding interface located around the entrance into the active site channel. In a, b, key residues are shown as sticks and surfaces of the lipoyl (a) and catalytic (b) domains are colored based on the electrostatic potential (values in kT/e). c Interaction of the dihydrolipoyllysine residue (cyan) with the residues in the active site channel at the interface of two catalytic domains (magenta and green, respectively). d Comparison of the active site of E. coli E2p (color code is the same as in b) with the structures of the complexes of Azotobacter vinelandii E2p with dihydrolipoate (PDB code 1eae, black) and oxidized CoA (PDB code 1ead, brown; only the catalytic His and Asn are shown), and Bos taurus E2b with CoA (PDB code 2ii4, light gray). Residue numbers correspond to E. coli E2p. e Cryo-EM map for the dihydrolipoyllysine residue.