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

The pyruvate dehydrogenase complex (PDHc) is a crucial metabolic enzyme complex that converts pyruvate to acetyl-coenzyme A, linking glycolysis to the citric acid cycle. Its importance is highlighted by its involvement in glucose homeostasis and its roles in various diseases, including obesity, type 2 diabetes, neurodegenerative disorders, and cancer. PDHc comprises three enzymatically active subunits: pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoyl dehydrogenase (E3). E2p, a multidomain protein, forms the structural core, binds other enzymes, and shuttles intermediates via lipoyl domains. While the overall function is understood, the precise molecular mechanism of this substrate shuttling has remained unclear. This study aimed to elucidate this mechanism through cryo-EM analysis of the native *E. coli* E2p core.

Extensive research has been conducted on PDHc, focusing on its structure and function. Previous studies have revealed the overall architecture of the complex, including the cubic arrangement of E2p subunits in bacteria and the roles of individual domains within E2p. However, the details of the interaction between lipoyl domains and the catalytic core, and how these interactions facilitate substrate channeling, have not been fully resolved. Many structural studies using X-ray crystallography and other methods have provided valuable insights into individual components and subcomplexes, yet a high-resolution structure of the complete, native complex in a physiologically relevant state was needed to fully understand substrate shuttling.

The researchers isolated the native PDHc from *E. coli* K12 cells grown in minimal media with succinate as the sole carbon source to obtain a resting state. The purity and integrity of the complex were verified using mass spectrometry and enzymatic assays. The presence of lipoamide modification was confirmed by Western blot and mass spectrometry. Cryo-EM analysis was performed on the isolated complex. Single-particle cryo-EM data was collected using both Talos Arctica and Titan Krios microscopes. CryoSPARC software was utilized for data processing, including particle picking, 2D and 3D classification, and refinement. The final 3D reconstruction of the E2p core reached a nominal resolution of 3.16 Å. Model building was performed using the X-ray structure of the catalytic domain (PDB 4n72) and the NMR structure of the lipoyl domain (PDB 1qjo) as initial models, followed by manual building and refinement using Phenix and Coot. Mass spectrometry was employed to analyze the lipoylation state of the complex, including the identification of lipoylated lysine residues and the proportion of acetylated and non-acetylated forms. Western blotting confirmed the presence of lipoic acid modifications.

The cryo-EM reconstruction revealed a cubic E2p core with octahedral symmetry, composed of trimers of catalytic domains. Each trimer had associated lipoyl domains, which appeared as protrusions in the map. The structure showed that the dihydrolipoyllysine residue is deeply embedded within the E2p active site channel. Detailed analysis of the interface between E2p trimers revealed an interaction area of 840 Ų stabilized by hydrophobic and polar interactions. The interaction between the lipoyl domain and the catalytic domain is characterized by an electrostatic network, with an acidic patch on the lipoyl domain interacting with a positive dipole on the catalytic domain helix H1 and other positively charged residues. Specific hydrogen bonds and van der Waals interactions between loops of the two domains were also identified. The dihydrolipoyllysine residue interacts primarily through hydrophobic interactions with residues in the active site channel, which is formed at the interface of two catalytic domains. The active site channel shows structural similarity to homologous structures in other E2 enzymes, while the conformation of the dihydrolipoyllysine differs slightly from that seen in other structures due to its attachment to the lipoyl domain and its flexibility. The structure suggests that *E. coli* E2p exists in an open-gate conformation in the resting state, binding the lipoyl domain-bound dihydrolipoamide in the absence of coenzyme A, unlike the substrate-mediated gating mechanism observed in some eukaryotic E2 enzymes.

This study provides the first high-resolution structure of the native *E. coli* E2p core, revealing the molecular details of lipoyl domain binding and substrate insertion. The electrostatic interactions between the lipoyl and catalytic domains likely provide a primary navigation mechanism for the substrate into the active site. The predominantly hydrophobic interactions of the dihydrolipoyllysine within the active site channel suggest a mechanism that is distinct from substrate-mediated gating. The open-gate conformation in the absence of CoA further supports this. These findings refine our understanding of the substrate channeling mechanism in PDHc and contribute to a broader understanding of multienzyme complex function. The conserved electrostatic interactions suggest that the mechanism may be common to E2 enzymes across different organisms.

This study presents the high-resolution cryo-EM structure of the native *E. coli* PDHc E2p core, resolving the interaction between lipoyl domains and the catalytic domain. The findings elucidate the mechanism of substrate insertion and suggest a conserved electrostatic navigation system among E2 enzymes. Future studies could investigate the dynamic interactions during catalysis and explore the structural adaptations in other PDHc variants and related complexes.

The study focused on the E2p core in a resting state. The structure may not fully represent the dynamic changes that occur during the catalytic cycle. Also, while the overall structure of the outer shell was observed at lower resolution, the individual components (E1p and E3) could not be unambiguously assigned due to flexibility and heterogeneity. The inability to definitively determine the oxidation state of the lipoyllysine in the resting state is another limitation.