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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.

SUBMITTER: Skerlova J

PROVIDER: S-EPMC8421416 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2+E3BP) core organisation: the 'addition' model (60+12) and the 'substitution' model (48+12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the 'substitution' model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.

Project description:The human pyruvate dehydrogenase complex (hPDC) is a large macromolecular machine, and its unique structural and functional properties make it a versatile target for manipulation aiming for the design of new types of artificial multienzyme cascades. However, model-based and hence systematic understanding of the structure-function relationship of this kind of complexes is yet poor. However, with new structure data, modeling techniques, and increasing computation power available, this shortfall is about to cease. Recently, we have built new atomistic models of E2/E3BP, the two subunits of the massive hPDC core. Here, we present developed coarse-grained models of the same, on the basis of which we built and simulated the full core of hPDC, which is so far the first simulation of the catalytic core of any member in the branched-chain α-keto acid dehydrogenase complex family. We explored the stability of two previously proposed substitutional models of hPDC core: 40E2+20E3BP and 48E2+12E3BP. Our molecular dynamics simulations showed a higher stability and sphericity for the second model. Our core's radius of gyration was found to be in strong agreement with previously published experimental dynamic light scattering (DLS) data. Finally, in the direction of our experimental effort to design active minimized complexes, we simulated C-terminal truncated E2/E3BP cores of different lengths, which clearly showed the instability of the core assembly and symmetry due to subunit separations. This correlated very well with the findings of our experimental investigations by analysis of DLS data for variable truncation lengths. The use of polarizable water and an increased total ion concentration did not lead to a substantially different initial stability of the truncated mutants compared to that of standard MARTINI water; however, a different rearrangement behavior of the fragments was observed. The obtained structure models will serve as a basis for full complex simulations in the future, providing the possibility for the model-assisted targeted manipulation of such a complex enzymatic system.

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

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