Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure

Nagy, Balázs; Polák, Martin; Ozohanics, Olivér; Zambo, Zsofia; Szabó, Eszter; Hubert, Agnes; Jordan, Frank; Nováček, Jiří; Ádám-Vizi, Vera; Ambrus, Attila

doi:10.1016/j.bbagen.2021.129889

Cited by 27 publications

(78 citation statements)

References 143 publications

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“…In the preparation of mouse brain mitochondria, FAD content was estimated as 65.0 ± 4.7 pmol FAD per milligram of protein. It has been shown recently that a fully assembled KGDHC contains 12 DLD subunits per assembled complex ( 57 ). Therefore, the calculated stoichiometry of KGDHC is equivalent to 5.4 ± 0.4 pmol KGDHC/mg of protein.…”

Section: Discussionmentioning

confidence: 99%

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Ansari

Yoval-Sánchez

Niatsetskaya

et al. 2021

Journal of Biological Chemistry

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Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 10 4 min −1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 10 3 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

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Section: Discussionmentioning

confidence: 99%

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Ansari

Yoval-Sánchez

Niatsetskaya

et al. 2021

Journal of Biological Chemistry

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“…Additional validation for the identity of these complexes is provided by the relative abundance of the component proteins in the MS data (Figure 1C). Of note, the pre-60S from a thermophilic eukaryote has not been structurally characterized to date; in addition, only an overexpressed, truncated version of the OGDHc core has been reported (Nagy et al, 2021), whereas here, the native asymmetric and cubic reconstruction of the hybrid OGDHc/BCKDHc core is communicated.…”

Section: Identification and Recovery Of Heterogeneous Cryo-em Structural Signatures Within A Single Cellular Fractionmentioning

confidence: 93%

Cryo-EM and artificial intelligence visualize endogenous protein community members

et al. 2022

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“…Some 15 years later, the molecular organization of the E. coli PDHc and OGDHc was reinvestigated by using cryo-electron tomography and it was demonstrated that the E1 and E3 components in these complexes are flexibly tethered ~11 nm away from the E2 core with no significant variations in the E2 core dimensions [105]. Recently, a 3D reconstruction of the human E2o core at 4.7Å resolution [35] and at 2.9Å resolution [115] was generated by single-particle cryo-EM. The EM reconstruction showed 24 C-terminal core domains grouped as eight trimers and positioned at each of the eight vertices of the cubic cage with octahedral symmetry [35,115].…”

Section: Application Of Cryo-em To Gain Insight Into the Architecture Of The 2-oxo Acid Dehydrogenase Complexesmentioning

confidence: 99%

“…Recently, a 3D reconstruction of the human E2o core at 4.7Å resolution [35] and at 2.9Å resolution [115] was generated by single-particle cryo-EM. The EM reconstruction showed 24 C-terminal core domains grouped as eight trimers and positioned at each of the eight vertices of the cubic cage with octahedral symmetry [35,115]. There was no electron density found for the other parts of the E2o, including the N-terminal lipoyl domain and much of the linker region, suggesting that those regions are highly flexible and dynamic [35,115].…”

Section: Application Of Cryo-em To Gain Insight Into the Architecture Of The 2-oxo Acid Dehydrogenase Complexesmentioning

confidence: 99%

“…The EM reconstruction showed 24 C-terminal core domains grouped as eight trimers and positioned at each of the eight vertices of the cubic cage with octahedral symmetry [35,115]. There was no electron density found for the other parts of the E2o, including the N-terminal lipoyl domain and much of the linker region, suggesting that those regions are highly flexible and dynamic [35,115]. The human E2o core structure generated was similar those reported earlier for the E. coli E2o core [47], the E. coli E2p core [52] and for the A. vinelandii E2p core [49], all obtained by X-ray crystallography.…”

Section: Application Of Cryo-em To Gain Insight Into the Architecture Of The 2-oxo Acid Dehydrogenase Complexesmentioning

confidence: 99%

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Toward an Understanding Of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxo Acid Dehydrogenase Complexes

Nemeria¹,

Zhang²,

Leandro³

et al. 2021

Preprint

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The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the TCA cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxo acid dehydrogenase complexes, their function and regulation.

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Structure of the dihydrolipoamide succinyltransferase (E2) component of the human alpha-ketoglutarate dehydrogenase complex (hKGDHc) revealed by cryo-EM and cross-linking mass spectrometry: Implications for the overall hKGDHc structure

Cited by 27 publications

References 143 publications

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Cryo-EM and artificial intelligence visualize endogenous protein community members

Toward an Understanding Of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxo Acid Dehydrogenase Complexes

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