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Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart

Abstract

Advancements in cross-linking mass spectrometry bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. In this study, we used isobaric quantitative protein interaction reporter (iqPIR) technology to compare the mitochondrial protein interactomes in healthy and failing murine hearts 4 weeks after transverse aortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondrial respiration; and conformational enrichment of the ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondrial remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.

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Fig. 1: Quantitation of mitochondrial protein interactome in failing hearts.
Fig. 2: Decreased interaction between NDUA4 and C6XB1 affects CIV activity in TAC.
Fig. 3: Active conformational states of ketone oxidation proteins enriched in TAC.
Fig. 4: Enrichment of an intermediate state of ADP/ATP carrier detected in TAC.

Data availability

Cross-linking data have been deposited at XlinkDB (http://xlinkdb.gs.washington.edu/xlinkdb/index.php) and are publicly available (network name: Caudal_iqPIR_TACsham_Bruce). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE70 partner repository with the dataset identifiers PXD027757 and PXD035622. The following publicly available files were included: PDB 5Z62, PDB 3DLX, PDB 3OXO, PDB 1OKC, PDB 6GCI, PDB 2LCK and UniProt P48962. Any additional information required to reanalyze the data reported in this paper is available from the lead contacts upon reasonable request. All other data supporting the findings in this study are included in the main article and associated files.

References

  1. Rath, S. et al. MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations. Nucleic Acids Res. 49, D1541–D1547 (2020).

    PubMed Central  Article  CAS  Google Scholar 

  2. Pagliarini, D. J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Qi, L. et al. Cryo-EM structure of the human mitochondrial translocase TIM22 complex. Cell Res. 31, 369–372 (2021).

    CAS  PubMed  Article  Google Scholar 

  4. Bridges, H. R. et al. Structure of inhibitor-bound mammalian complex I. Nat. Commun. 11, 5261 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. Gu, J. et al. Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1. Science 364, 1068 (2019).

    CAS  PubMed  Article  Google Scholar 

  6. Spikes, T. E., Montgomery, M. G. & Walker, J. E. Structure of the dimeric ATP synthase from bovine mitochondria. Proc. Natl Acad. Sci. USA 117, 23519 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Tucker, K. & Park, E. Cryo-EM structure of the mitochondrial protein-import channel TOM complex at near-atomic resolution. Nat. Struct. Mol. Biol. 26, 1158–1166 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Sinz, A. Crosslinking mass spectrometry goes in-tissue. Cell Syst. 6, 10–12 (2018).

    CAS  PubMed  Article  Google Scholar 

  9. Tang, X., Munske, G. R., Siems, W. F. & Bruce, J. E. Mass spectrometry identifiable cross-linking strategy for studying protein−protein interactions. Anal. Chem. 77, 311–318 (2005).

    CAS  PubMed  Article  Google Scholar 

  10. Schweppe, D. K. et al. Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry. Proc. Natl Acad. Sci. USA 114, 1732–1737 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Chavez, J. D. et al. Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus–plant interactions. J. Proteome Res. 11, 2968–2981 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Weisbrod, C. R. et al. In vivo protein interaction network identified with a novel real-time cross-linked peptide identification strategy. J. Proteome Res. 12, 1569–1579 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Chavez, J. D., Schweppe, D. K., Eng, J. K. & Bruce, J. E. In vivo conformational dynamics of Hsp90 and its interactors. Cell Chem. Biol. 23, 716–726 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Chavez, J. D. et al. Chemical crosslinking mass spectrometry analysis of protein conformations and supercomplexes in heart tissue. Cell Syst. 6, 136–141 (2018).

    CAS  PubMed  Article  Google Scholar 

  15. Ong, S.-E. et al. Stable isotope labeling by amino acids in cell culture, silac, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    CAS  PubMed  Article  Google Scholar 

  16. Chavez, J. D. Quantitative interactome analysis reveals a chemoresistant edgotype. Nat. Commun. 6, 7928 (2015).

    CAS  PubMed  Article  Google Scholar 

  17. Chavez, J. D., Keller, A., Zhou, B., Tian, R. & Bruce, J. E. Cellular interactome dynamics during paclitaxel treatment. Cell Rep. 29, 2371–2383 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Chavez, J. D., Keller, A., Mohr, J. P. & Bruce, J. E. Isobaric quantitative protein interaction reporter technology for comparative interactome studies. Anal. Chem. 92, 14094–14102 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Brown, D. A. et al. Mitochondrial function as a therapeutic target in heart failure. Nat. Rev. Cardiol. 14, 238–250 (2017).

    CAS  PubMed  Article  Google Scholar 

  20. Barth, E., Stämmler, G., Speiser, B. & Schaper, J. Ultrastructural quantitation of mitochondria and myofilaments in cardiac muscle from 10 different animal species including man. J. Mol. Cell. Cardiol. 24, 669–681 (1992).

    CAS  PubMed  Article  Google Scholar 

  21. Schweppe, D. K. et al. XLinkDB 2.0: integrated, large-scale structural analysis of protein crosslinking data. Bioinformatics 32, 2716–2718 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Broom, B. M. et al. A galaxy implementation of next-generation clustered heatmaps for interactive exploration of molecular profiling data. Cancer Res. 77, e23 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Zong, S. et al. Structure of the intact 14-subunit human cytochrome c oxidase. Cell Res. 28, 1026–1034 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Balsa, E. et al. NDUFA4 is a subunit of complex IV of the mammalian electron transport chain. Cell Metab. 16, 378–386 (2012).

    CAS  PubMed  Article  Google Scholar 

  25. Massa, V. et al. Severe infantile encephalomyopathy caused by a mutation in COX6B1, a nucleus-encoded subunit of cytochrome c oxidase. Am. J. Hum. Genet. 82, 1281–1289 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Abdulhag, U. N. et al. Mitochondrial complex IV deficiency, caused by mutated COX6B1, is associated with encephalomyopathy, hydrocephalus and cardiomyopathy. Eur. J. Hum. Genet. 23, 159–164 (2015).

    CAS  PubMed  Article  Google Scholar 

  27. Rosca, M. G. et al. Cardiac mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation. Cardiovasc. Res. 80, 30–39 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Murashige, D. et al. Comprehensive quantification of fuel use by the failing and nonfailing human heart. Science 370, 364–368 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  29. Aubert, G. et al. The failing heart relies on ketone bodies as a fuel. Circulation 133, 698–705 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Bedi, K. C. et al. Evidence for intramyocardial disruption of lipid metabolism and increased myocardial ketone utilization in advanced human heart failure. Circulation 133, 706–716 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Kolwicz, S. C., Airhart, S. & Tian, R. Ketones step to the plate. Circulation 133, 689–691 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  32. Uchihashi, M. et al. Cardiac-specific Bdh1 overexpression ameliorates oxidative stress and cardiac remodeling in pressure overload-induced heart failure. Circ. Heart. Fail. 10, e004417 (2017).

    CAS  PubMed  Article  Google Scholar 

  33. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Tammam, S. D., Rochet, J.-C. & Fraser, M. E. Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A. Biochemistry 46, 10852–10863 (2007).

    CAS  PubMed  Article  Google Scholar 

  35. Fraser, M. E., Hayakawa, K. & Brown, W. D. Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA. Biochemistry 49, 10319–10328 (2010).

    CAS  PubMed  Article  Google Scholar 

  36. Coker, S.-F. et al. The high-resolution structure of pig heart succinyl-CoA:3-oxoacid coenzyme A transferase. Acta Crystallogr. Biol. D Crystallogr. 66, 797–805 (2010).

    CAS  Article  Google Scholar 

  37. Shafqat, N. et al. A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency. J. Inherit. Metab. Dis. 36, 983–987 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302–2309 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Pebay-Peyroula, E. et al. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426, 39–44 (2003).

    CAS  PubMed  Article  Google Scholar 

  40. Ruprecht, J. J. et al. The molecular mechanism of transport by the mitochondrial ADP/ATP carrier. Cell 176, 435–447 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Karch, J. et al. Inhibition of mitochondrial permeability transition by deletion of the ANT family and CypD. Sci. Adv. 5, eaaw4597 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Bround, M. J., Bers, D. M. & Molkentin, J. D. A 20/20 view of ANT function in mitochondrial biology and necrotic cell death. J. Mol. Cell. Cardiol. 144, A3–A13 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Bullock, J. M. A., Thalassinos, K. & Topf, M. Jwalk and MNXL web server: model validation using restraints from crosslinking mass spectrometry. Bioinformatics 34, 3584–3585 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Mirdita, M. et al. ColabFold—making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Bertholet, A. M. et al. H+ transport is an integral function of the mitochondrial ADP/ATP carrier. Nature 571, 515–520 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Ramzan, R., Rhiel, A., Weber, P., Kadenbach, B. & Vogt, S. Reversible dimerization of cytochrome c oxidase regulates mitochondrial respiration. Mitochondrion 49, 149–155 (2019).

    CAS  PubMed  Article  Google Scholar 

  47. Kokoszka, J. E. et al. The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 427, 461–465 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Chavez, J. D. et al. Mitochondrial protein interaction landscape of SS-31. Proc. Natl Acad. Sci. USA 117, 15363 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Tarnavski, O. et al. Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies. Physiol. Genomics 16, 349–360 (2004).

    CAS  PubMed  Article  Google Scholar 

  50. Ritterhoff, J. et al. Metabolic remodeling promotes cardiac hypertrophy by directing glucose to aspartate biosynthesis. Circ. Res. 126, 182–196 (2020).

    CAS  PubMed  Article  Google Scholar 

  51. Thompson, A. et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003).

    CAS  PubMed  Article  Google Scholar 

  52. Ross, P.ÿL. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    CAS  PubMed  Article  Google Scholar 

  53. Chavez, J. D. et al. Systems structural biology measurements by in vivo cross-linking with mass spectrometry. Nat. Protoc. 14, 2318–2343 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

    CAS  PubMed  Article  Google Scholar 

  55. Rogers, G. W. et al. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS ONE 6, e21746 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. Grinblat, L., Pacheco Bolaños, L. F. & Stoppani, A. O. Decreased rate of ketone-body oxidation and decreased activity of d-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats. Biochem. J. 240, 49–56 (1986).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Williamson, D. H., Bates, M. W., Page, M. A. & Krebs, H. A. Activities of enzymes involved in acetoacetate utilization in adult mammalian tissues. Biochem. J. 121, 41–47 (1971).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Brahma, M. K. et al. Increased glucose availability attenuates myocardial ketone body utilization. J. Am. Heart Assoc. 9, e013039 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. Berardi, M. J., Shih, W. M., Harrison, S. C. & Chou, J. J. Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching. Nature 476, 109–113 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. Keller, A., Chavez, J. D., Tang, X. & Bruce, J. E. Leveraging the entirety of the Protein Data Bank to enable improved structure prediction based on cross-link data. J. Proteome Res. 20, 1087–1095 (2021).

    CAS  PubMed  Article  Google Scholar 

  61. Spinazzi, M., Casarin, A., Pertegato, V., Salviati, L. & Angelini, C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat. Protoc. 7, 1235–1246 (2012).

    CAS  PubMed  Article  Google Scholar 

  62. Chen, Z. et al. Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes. Wellcome Open Res. 1, 5 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  63. Chen, Z. A. & Rappsilber, J. Protein dynamics in solution by quantitative crosslinking/mass spectrometry. Trends Biochem. Sci. 43, 908–920 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  64. Keller, A., Eng, J., Zhang, N., Li, X.-J. & Aebersold, R. A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol. Syst. Biol. 1, 2005.0017 (2005).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  65. Mohr, J. P., Perumalla, P., Chavez, J. D., Eng, J. K. & Bruce, J. E. Mango: a general tool for collision induced dissociation-cleavable cross-linked peptide identification. Anal. Chem. 90, 6028–6034 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. Chavez, J. D., Weisbrod, C. R., Zheng, C., Eng, J. K. & Bruce, J. E. Protein interactions, post-translational modifications and topologies in human cells. Mol. Cell. Proteomics 12, 1451–1467 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  67. Calvo, S. E., Clauser, K. R. & Mootha, V. K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  68. Keller, A., Chavez, J. D. & Bruce, J. E. Increased sensitivity with automated validation of XL-MS cleavable peptide crosslinks. Bioinformatics 35, 895–897 (2018).

    PubMed Central  Article  CAS  Google Scholar 

  69. Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications Made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002).

    CAS  PubMed  Article  Google Scholar 

  70. Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2018).

    PubMed Central  Article  CAS  Google Scholar 

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Acknowledgements

We thank all Tian and Bruce laboratory members for their thoughtful discussions and support. We thank the University of Washington Proteomics Resource for advice and helpful discussions. We thank Y.-W. A. Hsu for assistance with the animal models. We thank J. Ritterhoff and F. Drees for their technical support and guidance. This work was supported, in part, by National Institutes of Health (NIH) grants HL110349, HL129510 and HL142628 (to R.T.); HL144778, GM097112, GM086688 and R35GM136255 (to J.E.B); American Heart Association (AHA) Predoctoral Fellowship 20PRE35120126 (to A.C.); AHA Postdoctoral Fellowship 18POST33990352 (to B.Z.); China Scholarship Council Fellowship 202006320416 (to H.C.); and NIH 2T32DK007247-41 and AHA Career Development Award 930223 (to M.A.W).

Author information

Authors and Affiliations

Authors

Contributions

A.C., X.T., J.D.C., R.T. and J.E.B. designed the experiments. A.C., X.T., J.D.C., A.K., M.A.W., R.T. and J.E.B. wrote the manuscript. A.C., X.T., R.T. and J.E.B. edited the manuscript. A.C., X.T., J.D.C. and A.K. performed formal analysis. A.C., B.Z. and M.A.W. performed animal experiments. A.C. and J.D.C. performed cross-linking experiments. J.D.C. performed protein preparation. J.D.C., X.T. and A.K. performed mass spectrometry raw data acquisition and processing. A.K. developed computational tools to support structural protein analysis and cross-linking quantitation. J.P.M. and A.A.B. performed cross-linking analysis and structural modeling. O.V. and H.C. performed animal surgeries. R.T. and J.E.B. supervised the project.

Corresponding authors

Correspondence to Rong Tian or James E. Bruce.

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Nature Cardiovascular Research thanks Claudio Iacobucci, Martin Steinegger, Daniel Kelly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 (a-b) Left ventricle internal dimension and wall thickness in TAC and Sham groups determined by echocardiography four weeks post-surgery.

(c-d) Lung and liver edema (wet weight/dry weight in mg) measured at tissue harvest. (e) Quantitation of mean cross-link (XL) ratio vs mean dead-end (DE) ratio for each cross-linked peptide pair statistically changed in TAC in at least 4/6 biological pairs (Log2 TAC/Sham). The sum of the Mean DE ratio for Protein A and Protein B is shown to account for cross-links between two different proteins. (f) Interaction network of lysine residues (black nodes) connected by observed cross-links (edges). Edges are colored according to increasing (red) and decreasing (blue) quantitation of statistically significant subset of cross-links (Log2 TAC/Sham) shown by color scale. For (a-d), all data are n=12 animals, AVG+/-SEM, *denotes p<0.05 by unpaired, two-tailed Student’s t-test.

Extended Data Fig. 2 (a) Structural insight into CX6B1 R20 forming salt-bridges at NDUA4-CX6B1 interface.

R20 side chain (green) forms a salt bridge with CX6B1 D16 (green) and NDUA4 D60 (black), which pinpoints an interface necessary for the stability of CIV. Side-chains of R20, D16, and NDUA4 D60 (partially resolved) are depicted in stick representation. Cross-linked lysine sidechains are shown as yellow (CX6B1) or orange (NDUA4) spheres. (b) Cytochrome C oxidase enzymatic activity assay in tissue homogenates from TAC and Sham hearts, normalized to Citrate Synthase activity. N=4 animals, AVG+/-SEM, *denotes p<0.05 by unpaired, two-tailed Student’s t-test. (c) Table summarizing the mean cross-linking ratio and DE ratio for cross-linked peptide pairs, including values obtained across biological replicates for cross-links in ADT isoforms. (d) Representative Blue Native-PAGE (BN-PAGE) analysis of mitochondria isolated from Sham and TAC groups. Coomassie stain (left) for total protein loading, In-gel CIV activity stain (middle), with CI activity stain overlay (right). Gels were run in duplicates. (e) Representative BN-PAGE immunoblot of NDUA4 containing SCs from digitonin solubilized isolated mitochondria. Blots were run in duplicates. (f) Representative BN-PAGE immunoblot of NDUA4 containing SCs from DDM solubilized isolated mitochondria. Blots were run in duplicates. (g) Cross-linked peptide pairs (yellow lysine side chains) mapped onto the M-state conformation of ADT1 (PDB: 6GCI). Salt bridges between K96-D196 and K199-D292 contribute to the gating mechanism, which closes the M-state to the IMS and would make lysines unavailable for cross-linking. Aspartic acid sidechains are shown in magenta. (h) Cross-linked peptide pairs (yellow lysine side chains) mapped onto the C-state conformation of bovine ADT1 (PDB: 1OKC). K33 and D231 are known to form a salt bridge that stabilizes the C-state and would make K33 unavailable for cross-linking. Aspartic acid sidechains are shown in magenta.

Source data

Extended Data Fig. 3 (a) AlphaFold-predicted BDH1_MOUSE structure.

Crosslinked residues were indicated in yellow spheres, and crosslinks shown in red lines mean they were increased in TAC samples. (b) The pLDDT plot of the predicted BDH_mouse structure. (c) Alignment of apo (grey, PDB: 3OXO chain A) and substrate-bound (yellow, PDB: 3OXO chain E) monomers of porcine SCOT1. CoA is colored in magenta and bound to the active site. Lysine sidechains are shown in stick representation. Alignment depicts the structural differences between the dynamic C-terminal domain and the static N-terminal domain during substrate-binding. A close-up view specifies cross-linked lysines (K418 and K421). (d) Ketone-driven oxygen consumption rate (OCR) of mitochondria isolated from TAC and Sham hearts. Baseline OCR (State 1) was measured, followed by sequential injections of β-hydroxybutyrate/malate (State 2), ADP (State 3), Oligomycin (State 4μ), and FCCP (FCCPmax). N=6 animals, AVG+/-SEM, *denotes p<0.05 by unpaired, two-tailed Student’s t-test.

Supplementary information

Reporting Summary

Supplementary Video 1

Cross-linking determines ADT1 conformational states

Supplementary Table

Supplementary Data 1: log2 ratios of non-redundant peptide pairs across six biological replicates and their mean log2 ratios showed significant changes between TAC and Sham samples and DE mean log2 ratios of the corresponding proteins. Supplementary Data 2: LFQ of mitochondrial proteins from six biological replicates of TAC and Sham samples and combined log2 ratios of TAC/Sham was reported. Supplementary Data 3: R2 values of pairwise linear regression of six pairs of biological replicates and mean R2 values of FF and FR regression. Supplementary Data 6: log2 ratios of residue pairs across six biological replicates and their mean log2 ratios for each residue pair with P value and 95% confidence level and several distinct peptide pairs reported. Supplementary Data 7: ANOVA test of the quantitation of the same residue pair generated from fully-cleaved peptide pairs and its corresponding missed-cleaved peptide pairs

Supplementary Data 4

SCOT1 octamer structure file generated by superimposition of AlphaFold monomer structures onto 3OXO to provide structural data on missing regions containing K296

Supplementary Data 5

AlphaFold model structure of ADT1 open channel consistent with increased cross-link levels quantified in TAC hearts

Source data

Source Data Extended Data Fig. 2

Uncropped gels for Extended Data Fig. 2c–e

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Caudal, A., Tang, X., Chavez, J.D. et al. Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart. Nat Cardiovasc Res 1, 855–866 (2022). https://doi.org/10.1038/s44161-022-00127-4

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