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OPA1 helical structures give perspective to mitochondrial dysfunction

Nature volume 620, pages 1109–1116 (2023)Cite this article

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Abstract

Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60–80% of cases1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria2. Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes3. Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide. These helical assemblies organize into densely packed protein rungs with minimal inter-rung connectivity, and exhibit nucleotide-dependent dimerization of the GTPase domains—a hallmark of the dynamin superfamily of proteins4. OPA1 also contains several unique secondary structures in the paddle domain that strengthen its membrane association, including membrane-inserting helices. The structural features identified in this study shed light on the effects of pathogenic point mutations on protein folding, inter-protein assembly and membrane interactions. Furthermore, mutations that disrupt the assembly interfaces and membrane binding of OPA1 cause mitochondrial fragmentation in cell-based assays, providing evidence of the biological relevance of these interactions.

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Fig. 1: Cryo-EM structure of s-OPA1 in the presence of GDP-AlFx, highlighting oligomeric interfaces within the helical assembly.
Fig. 2: Interfaces involved in s-OPA1 helical assembly.
Fig. 3: Comparison of s-OPA1 dimers and assembly in apo and GDP-AlFx-bound states.
Fig. 4: Examining the functional effect of interface and membrane-binding OPA1 mutants.
Fig. 5: Model of IMM fusion by s-OPA1.

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Data availability

Structural data supporting findings in this study have been deposited in the PDB and the Electron Microscopy Data Bank (EMDB). The accession codes of the cryo-EM. maps and accompanying atomic models are provided for the following: (1) s-OPA1 GDP-AlFx:dimer (PDB ID: 8EEW) and s-OPA1 GDP-AlFx helical assembly (EMD-28063); (2) s-OPA1 apo:dimer (PDB ID: 8EF7) and s-OPA1 apo helical assembly (EMD-28074); (3) s-OPA1 GDP-AlFx:tetramer (PDB ID: 8EFF); (4) s-OPA1 apo:tetramer (PDB ID: 8EFS); (5) s-OPA1 GDP-AlFx:9-dimers (PDB ID: 8EFR); and (6) s-OPA1 apo:9-dimers (PDB ID: 8EFT). Additional submitted maps include: (1) z-clipped s-OPA1 GDP-AlFx helical assembly (EMD-40192); (2) z-clipped s-OPA1 apo helical assembly (EMD-40193); (3) s-OPA1 GDP-AlFx dimer (interface 1–3) (EMD-40200); (4) s-OPA1 apo dimer (interface 1–3) (EMD-40202); (5) s-OPA1 GDP-AlFx interface 4 (EMD-40210); (6) s-OPA1 apo interface 4 (EMD-40211); (7) s-OPA1 GDP-AlFx interface 7 (EMD-40212); (8) s-OPA1 apo interface 7 (EMD-40213); (9) s-OPA1 GDP-AlFx stalk:paddles (interface 1–3) (EMD-40203); (10) s-OPA1 apo stalk:paddles (interface 1–3) (EMD-40204); (11) s-OPA1 GDP-AlFx interface 5–6 (EMD-40214); (12) s-OPA1 GDP-AlFx interface 8 (EMD-40198); (13) s-OPA1 GDP-AlFx monomer A (EMD-40215); (14) s-OPA1 GDP-AlFx monomer B (EMD-40216); (15) s-OPA1 GDP-AlFx helix, N-terminal mask (EMD-40197); and (16) s-OPA1 GDP-AlFx membrane-inserting loop (Pα2):cardiolipin monolayer (EMD-40217). Plasmids from cell experiments (Supplementary Table 3) have been deposited in Addgene. Raw images for Extended Data Figs. 2b and 10c are available in Supplementary Fig. 1. Additional data used for comparisons in this study can be accessed through the PDB or AlphaFold: PDB ID: 6JTG, Alphafold ID: AF-O60313-F, PDB ID: 6QL4, PDB ID: 6JSJ, PDB ID:6RZV, PDB ID: 6RZW, PDB ID: 3ZYC, PDB ID:6DLV. Source data are provided with this paper.

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References

  1. Le Roux, B. et al. OPA1: 516 unique variants and 831 patients registered in an updated centralized Variome database. Orphanet J. Rare Dis. 14, 214 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Gao, S. & Hu, J. Mitochondrial fusion: the machineries in and out. Trends Cell Biol. 31, 62–74 (2021).

    Article  CAS  PubMed  Google Scholar 

  3. Del Dotto, V. & Carelli, V. Dominant optic atrophy (DOA): modeling the kaleidoscopic roles of OPA1 in mitochondrial homeostasis. Front. Neurol. 12, 681326 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Jimah, J. R. & Hinshaw, J. E. Structural insights into the mechanism of dynamin superfamily proteins. Trends Cell Biol. 29, 257–273 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. Chan, D. C. Mitochondrial fusion and fission in mammals. Annu. Rev. Cell Dev. Biol. 22, 79–99 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Horvath, S. E. & Daum, G. Lipids of mitochondria. Prog. Lipid Res. 52, 590–614 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Mishra, P. & Chan, D. C. Mitochondrial dynamics and inheritance during cell division, development and disease. Nat. Rev. Mol. Cell Biol. 15, 634–646 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Francy, C. A., Clinton, R. W., Fröhlich, C., Murphy, C. & Mears, J. A. Cryo-EM studies of Drp1 reveal cardiolipin interactions that activate the helical oligomer. Sci. Rep. 7, 10744 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  9. Macdonald, P. J. et al. A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission. Mol. Biol. Cell 25, 1905–1915 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Stepanyants, N. et al. Cardiolipin’s propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission. Mol. Biol. Cell 26, 3104–3116 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fröhlich, C. et al. Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein. EMBO J. 32, 1280–1292 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Song, Z., Ghochani, M., McCaffery, J. M., Frey, T. G. & Chan, D. C. Mitofusins and OPA1 mediate sequential steps in mitochondrial membrane fusion. Mol. Biol. Cell 20, 3525–3532 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, Y. & Chan, D. C. New insights into mitochondrial fusion. FEBS Lett. 581, 2168–2173 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Del Dotto, V., Fogazza, M., Carelli, V., Rugolo, M. & Zanna, C. Eight human OPA1 isoforms, long and short: what are they for? Biochim. Biophys. Acta Bioenerg. 1859, 263–269 (2018).

    Article  PubMed  Google Scholar 

  15. Lee, H. & Yoon, Y. Mitochondrial membrane dynamics–functional positioning of OPA1. Antioxidants 7, 186 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Anand, R. et al. The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission. J. Cell Biol. 204, 919–929 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Landes, T. et al. OPA1 (dys)functions. Semin. Cell Dev. Biol. 21, 593–598 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Weisschuh, N. et al. Mutation spectrum of the OPA1 gene in a large cohort of patients with suspected dominant optic atrophy: identification and classification of 48 novel variants. PLoS One 16, e0253987 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li, D., Wang, J., Jin, Z. & Zhang, Z. Structural and evolutionary characteristics of dynamin-related GTPase OPA1. PeerJ 7, e7285 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Ban, T., Heymann, J. A., Song, Z., Hinshaw, J. E. & Chan, D. C. OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation. Hum. Mol. Genet. 19, 2113–2122 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yapa, N. M. B., Lisnyak, V., Reljic, B. & Ryan, M. T. Mitochondrial dynamics in health and disease. FEBS Lett. 595, 1184–1204 (2021).

    Article  CAS  PubMed  Google Scholar 

  22. Zhang, D. et al. Cryo-EM structures of S-OPA1 reveal its interactions with membrane and changes upon nucleotide binding. eLife 9, e50294 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yu, C. et al. Structural insights into G domain dimerization and pathogenic mutation of OPA1. J. Cell Biol. 219, e201907098 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Wang, R. et al. Identification of new OPA1 cleavage site reveals that short isoforms regulate mitochondrial fusion. Mol. Biol. Cell 32, 157–168 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ge, Y. et al. Two forms of Opa1 cooperate to complete fusion of the mitochondrial inner-membrane. eLife 9, e50973 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Faelber, K. et al. Structure and assembly of the mitochondrial membrane remodelling GTPase Mgm1. Nature 571, 429–433 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yan, L. et al. Structural analysis of a trimeric assembly of the mitochondrial dynamin-like GTPase Mgm1. Proc. Natl Acad. Sci. USA 117, 4061–4070 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Faelber, K. et al. Crystal structure of nucleotide-free dynamin. Nature 477, 556–560 (2011).

    Article  ADS  CAS  PubMed  Google Scholar 

  29. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. Zhong, E. D., Bepler, T., Berger, B. & Davis, J. H. CryoDRGN: reconstruction of heterogeneous cryo-EM structures using neural networks. Nat. Methods 18, 176–185 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jimah, J. R. & Hinshaw, J. E. Structural insights into the mechanism of dynamin superfamily proteins. Trends Cell Biol. 29, 257–273 (2019).

    Article  CAS  PubMed  Google Scholar 

  32. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Chappie, J. S. et al. A pseudoatomic model of the dynamin polymer identifies a hydrolysis-dependent powerstroke. Cell 147, 209–222 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chappie, J. S., Acharya, S., Leonard, M., Schmid, S. L. & Dyda, F. G domain dimerization controls dynamin’s assembly-stimulated GTPase activity. Nature 465, 435–440 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Allen, K. N., Entova, S., Ray, L. C. & Imperiali, B. Monotopic membrane proteins join the fold. Trends Biochem. Sci. 44, 7–20 (2019).

    Article  CAS  PubMed  Google Scholar 

  36. Prinz, W. A. & Hinshaw, J. E. Membrane-bending proteins. Crit. Rev. Biochem. Mol. Biol. 44, 278–291 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sharma, K. D., Heberle, F. A. & Waxham, M. N. Visualizing lipid membrane structure with cryo-EM: past, present, and future. Emerg. Top. Life Sci. 7, 55–65 (2023).

    Article  CAS  PubMed  Google Scholar 

  38. Biou, V. Lipid–membrane protein interaction visualised by cryo-EM: a review. Biochim. Biophys. Acta Biomembr. 1865, 184068 (2023).

    Article  CAS  PubMed  Google Scholar 

  39. Hedger, G. et al. Lipid-loving ANTs: molecular simulations of cardiolipin interactions and the organization of the adenine nucleotide translocase in model mitochondrial membranes. Biochemistry 55, 6238–6249 (2016).

    Article  CAS  PubMed  Google Scholar 

  40. Corey, R. A. et al. Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins. Sci. Adv. 7, eabh2217 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. Planas-Iglesias, J. et al. Cardiolipin interactions with proteins. Biophys. J. 109, 1282–1294 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Renner, L. D. & Weibel, D. B. Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes. Proc. Natl Acad. Sci. USA 108, 6264–6269 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Glancy, B., Kim, Y., Katti, P. & Willingham, T. B. The functional impact of mitochondrial structure across subcellular scales. Front. Physiol. 11, 541040 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Yang, Z. et al. Mitochondrial membrane remodeling. Front. Bioeng. Biotechnol. 9, 786806 (2022).

    Article  MathSciNet  PubMed  PubMed Central  Google Scholar 

  45. Liu, X., Weaver, D., Shirihai, O. & Hajnóczky, G. Mitochondrial ‘kiss-and-run’: interplay between mitochondrial motility and fusion–fission dynamics. EMBO J. 28, 3074–3089 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Mears, J. A. et al. Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission. Nat. Struct. Mol. Biol. 18, 20–26 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Ge, Y., Boopathy, S., Nguyen, T. H., Lugo, C. M. & Chao, L. H. Absence of cardiolipin from the outer leaflet of a mitochondrial inner membrane mimic restricts Opa1-mediated fusion. Front. Mol. Biosci. 8, 769135 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ban, T. et al. Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin. Nat. Cell Biol. 19, 856–863 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Ban, T., Kohno, H., Ishihara, T. & Ishihara, N. Relationship between OPA1 and cardiolipin in mitochondrial inner-membrane fusion. Biochim. Biophys. Acta Bioenerg. 1859, 951–957 (2018).

    Article  CAS  PubMed  Google Scholar 

  50. Osman, C., Voelker, D. R. & Langer, T. Making heads or tails of phospholipids in mitochondria. J. Cell Biol. 192, 7–16 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Beltrán-Heredia, E. et al. Membrane curvature induces cardiolipin sorting. Commun. Biol. 2, 225 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Rujiviphat, J. et al. Mitochondrial genome maintenance 1 (Mgm1) protein alters membrane topology and promotes local membrane bending. J. Mol. Biol. 427, 2599–2609 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. DeVay, R. M. et al. Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion. J. Cell Biol. 186, 793–803 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Joardar, A., Pattnaik, G. P. & Chakraborty, H. Mechanism of membrane fusion: interplay of lipid and peptide. J. Membr. Biol. 255, 211–224 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Frohman, M. A. Role of mitochondrial lipids in guiding fission and fusion. J. Mol. Med. 93, 263–269 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Paradies, G., Paradies, V., Ruggiero, F. M. & Petrosillo, G. Role of cardiolipin in mitochondrial function and dynamics in health and disease: molecular and pharmacological aspects. Cells 8, 728 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Guo, Y. et al. Visualizing Intracellular organelle and cytoskeletal interactions at nanoscale resolution on millisecond timescales. Cell 175, 1430–1442 (2018).

    Article  CAS  PubMed  Google Scholar 

  58. Leonard, M., Song, B. D., Ramachandran, R. & Schmid, S. L. Robust colorimetric assays for dynamin’s basal and stimulated GTPase activities. Methods Enzymol. 404, 490–503 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Diaz, R., Rice, W. J. & Stokes, D. L. Fourier–Bessel reconstruction of helical assemblies. Methods Enzymol. 482, 131–165 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  65. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  66. He, S. & Scheres, S. H. W. Helical reconstruction in RELION. J. Struct. Biol. 198, 163–176 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  69. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  71. DiMaio, F. Advances in Rosetta structure prediction for difficult molecular-replacement problems. Acta Crystallogr. D 69, 2202–2208 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Wang, R. Y. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, e17219 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  74. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 50, W276–W279 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016).

    Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the NIDDK and NHLBI NIH Intramural Research Program. The work used the NIH Multi-Institute Cryo-EM Facility (MICEF), the NIDDK Cryo-EM core facility, the NHLBI light microscopy core facility and the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We thank D. A. Nyenhuis, H. Wang, Y. Cui, H. Lei, A. D. Kehr and H. W. Breen for technical support and computing assistance.

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Author notes

  1. Abigail E. Stanton

    Present address: Molecular Biology Department, Princeton University, Princeton, NJ, USA

Authors and Affiliations

  1. Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA

    Sarah B. Nyenhuis, Abigail E. Stanton, Bertram Canagarajah & Jenny E. Hinshaw

  2. Light Microscopy Facility, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

    Xufeng Wu

  3. Protein Expression Facility, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

    Marie-Paule Strub

  4. Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

    Yang-In Yim & John A. Hammer

  5. Electron Microscopy Core, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

    Valentina Baena & Zulfeqhar A. Syed

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Contributions

S.B.N. and J.E.H. designed the research. S.B.N. and A.E.S. prepared protein samples. A.E.S. performed the GTPase assay. S.B.N. collected cryo-EM data. S.B.N., B.C. and J.E.H. processed and analysed the data. S.B.N., X.W. and J.A.H. designed, performed and analysed cell-based assays. M.-P.S. and S.B.N. generated all constructs for cell-based assays. Y.I-Y. grew and maintained cells for cell-based assays and performed western blots. V.B. and Z.A.S. performed FIB-SEM and TEM. S.B.N. and J.E.H. wrote the paper. All authors were asked to comment on the manuscript.

Corresponding author

Correspondence to Jenny E. Hinshaw.

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The authors declare no competing interests.

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Nature thanks Thomas Langer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Helical assemblies of s-OPA1 coating cardiolipin-containing membranes with and without GDP-AlFx.

a, Left, top view of s-OPA1 with GDP-AlFx bound, with 38.2 nm outer diameter and a 9.4 nm inner lumen diameter of the membrane. Centre, side view of the helical map with 10 modelled tetramers which define a single helical turn, coloured by domain (see Fig. 1). Right, a central slice of the helix is shown in comparison to a 2D classification generated in RELION (right corner). A radial profile plot is shown above the sliced helix. b, Left, top view of apo s-OPA1, with 40.5 nm outer diameter and a 9.5 nm inner lumen diameter of the membrane. Centre, side view of the helical map with 10 tetramers which define a single turn, coloured as in a. Right, a central slice of the helix is shown in comparison to a corresponding apo 2D classification generated in RELION (right corner). A radial profile plot is shown above the sliced helix. c, A z-clipped refinement focusing on the central around 30% of the GDP-AlFx-bound helix is coloured by domain, shown as a top view (left), side view (centre) and centrally sliced (right) with a single s-OPA1 dimer highlighted in darker hues. d, Domains of s-OPA1, moving from the outer diameter of the GDP-AlFx helix toward the membrane bilayer. Left, GTPase domains and BSE regions coloured green and pink, respectively. Centre, stalks shown in blue. Right, paddles coloured orange. e,f, same as c,d for the apo helix. g, s-OPA1 GDP-AlFx map with density coluored by domain as in a and refined in cryoSPARC without dynamic masking. Insets highlight the N terminus, coloured grey.

Extended Data Fig. 2 Workflow for determining s-OPA1 helical assembly and corresponding local refinements using cryoSPARC.

a, Sample preparation workflow for s-OPA1 purification, GTPase activity assay, and tube formation. Images for the GDP-AlFx and apo states were collected on a Titan Krios and a Glacios, respectively. GTPase activity data are presented as mean values ± s.d. of n = 3 independent experiments. b, Left, s-OPA1 tube diameters for both the apo (orange) and GDP-AlFx (blue) states. Asterisks mark diameters used for cryo-EM image processing. Right, Sup/Pellet assay showing GTP-mediated protein disassembly from membrane. SDS–PAGE gel (top) of Sup/Pellet assay with protein alone (*), and s-OPA1 tubes before (Apo) and after GTP addition (GTP). Bar graph (bottom) of Sup/Pellet assay with supernatant (blue) and pellet (orange) for the same conditions (data are presented as mean values ± s.d. of n = 3 independent experiments). c, Outline of data processing. Each dataset followed a standard workflow including patch motion correction and patch CTF correction. Segments of tubes were then manually selected, 2D classified to generate templates to trace the particles, extracted and curated to a 6 Å CTF cut-off. Particles were 2D classified and particles of similar identity and diameter were selected and aligned through ab initio modelling. The selected ab initio models were then refined with non-uniform helical refinement. For GDP-AlFx tubes, helical symmetry was determined using a combination of Fourier and real-space analysis using HELIXPLORER and RELION60,62,63. For Apo tubes, symmetry parameters were determined in cryoSPARC. Each set of parameters was examined with the cryoSPARC helical search tool29,65. To assess the interfaces of each OPA1 in the assembly, local refinements were performed by first symmetry expanding the particles in the helical refinements. Areas of interest were masked in chimera. Masks were then expanded and padded within cryoSPARC for particle subtraction, followed by non-uniform local refinements. The resulting maps were used to build the model of s-OPA1, which was generated using Coot, Rosetta, and PHENIX69,71,74.

Source data

Extended Data Fig. 3 Closer examination of the stalk and paddle interfaces within the s-OPA1 dimer.

a, A side view of a GDP-AlFx s-OPA1 dimer fit into a locally refined map. Monomers coloured by domain (see Fig. 1). Chain A is coloured a dark hue and chain B a light hue. The interlocked stalk interface 1 is shown above and paddle–paddle interface 3, where the paddle is rotated 180° showing the membrane-facing region, is displayed below. Insets: interface 4 between the paddle hinges and interface 7 between the inter-rung paddles are highlighted. b, Top view of isolated interfaces for the s-OPA1 stalk and paddle regions. The full dimeric interface between the stalks and paddles is shown fit into the locally refined density (centre) and coloured as in a. Interface 2 between the stalk of an s-OPA1 monomer and the paddle of the second monomer is shown for the A- to B-chain (top) and the B- to A-chain (bottom). c, A representative fit of each domain of the s-OPA1 model into the map density. d,e, Same as a,b for the apo dimer.

Extended Data Fig. 4 Interfaces involved in s-OPA1 GDP-AlFx helical assembly.

a, Left, the s-OPA1 dimer with its three interfaces labelled. Monomers coloured by domain (see Fig. 1). Chain A is coloured a dark hue and chain B a light hue. Top and side views of the helical map with a single dimer highlighted shown in top corners of dimer. Right, magnified views of each isolated interface with buried residues shown as sticks. b–g, s-OPA1 interfaces involved in higher-order assembly. In each case, colours are as in a. Chains of each s-OPA1 dimer not involved in the interface are greyed. Top corners, s-OPA1 in the context of the full helical map. Insets show residues buried in each interface as sticks. b, Two dimers of s-OPA1 assemble into a tetramer through interface 4. c, In the presence of GDP-AlFx, GTPase domains dimerize between dimer-1 A-chain and dimer-2 B-chain and form interface 5. d, In the presence of GDP-AlFx, interface 6 forms by a GTPase domain–stalk interaction between dimers A-chains (left) B-chains (right). e, Between rungs, interface 7 involves paddles of dimer-1 A-chain and dimer-2 B-chains. f, Another inter-rung interface, interface 8, appears between two N-terminal coiled-coils (purple) of dimer-1 A-chain and dimer-2 B-chain. g, Interface 9 is an N-terminal:BSE dimer interface between dimers A-chains (left) B-chains (right). Interfaces were determined using PDBePISA (ref. 32).

Extended Data Fig. 5 Comparison between s-OPA1 and dynamin stalk domain and GTPase dimer.

a, s-OPA1 stalk (blue) is kinked 30° relative to dynamin-1 stalk (PDB ID: 6DLV, white). b, GTPase dimer (green) highlighted in helical map. Inset, GTPase domain dimer (interface 5) with monomers coloured by domain (see Fig. 1). Chain A is coloured a dark hue and chain B a light hue. The crystal dimer (PDB ID: 6JTG, white) is aligned to our model. c, Our s-OPA1 model versus the 6JTG dimer, coloured as in a from the top (left) and two side views (centre, right) to highlight differences in dimer conformation. The BSE domains of both chains swing to accommodate the angular difference of hinge 1 connecting the stalks, resulting in the A- and B-chains fitting with slight asymmetry compared to the crystal structure. Interface 5 is labelled and circled. d, Our s-OPA1 model versus the dynamin-1 (PDB ID: 3ZYC) dimer with GMPPCP bound, coloured as in a from the top (left) and two side views (centre, right) to highlight differences in dimer conformation. The orientational difference at hinge 2 in the BSE domains is expected from the powerstroke model for DSPs.

Extended Data Fig. 6 Human s-OPA1 compared to s-MGM1 and enhanced s-OPA1 membrane binding.

a, Sequence comparison between s-OPA1 and s-MGM1 (ref. 26) (grey). s-OPA1 is coloured by domain (see Fig. 1). b, Overlay of s-OPA1 with s-MGM1 (grey) (PDB ID: 6QL4) highlighting similar domains. s-OPA1 domains are coloured according to a. Unique regions in s-MGM1 and s-OPA1 are highlighted: kinked stalks, paddle domains, and the stalk–paddle hinge disulfide bond. Inset shows a 180° rotation of the zoomed paddle tip highlighting differences in secondary structure and domain angle. Top left, s-OPA1 monomer highlighted in helical map. Helices are labelled as in Supplementary Fig. 2. c, A 90° rotation of s-OPA1 and s-MGM1 (grey), with an s-OPA1 monomer coloured in helical map, top left. The membrane is drawn in grey highlighting increased s-OPA1 membrane insertion, the membrane-inserting loop (Pα2) and Pα6 and closer OPA1 paddle-tip membrane association (inset). d, Table comparing structural features and sequences between OPA1 and MGM1. Positively charged residues are blue and aromatics are orange. e, Locally refined map of the bilayer outer leaflet with proposed cardiolipin density proximal to the Pα2 helix. s-OPA1 paddles are coloured as in a, cardiolipin is coloured magenta, unidentified lipids are white. To orient, a cylinder representing the membrane with a box representing the refined region appear, top left. f, A view of the map, rotated 180° from e. g, Three views of cardiolipin modelled into the map. h, s-OPA1 paddle with a possible cardiolipin orientation shown (sticks and surface views). Membrane-inserting, positively charged (blue) and aromatic (white) residues appear (sticks). A representative cylinder with the boxed region (top left). i, A 90° rotation of h. j, A magnified view of the s-OPA1 and two cardiolipin models (dark and light magenta) highlighting potential residues (sticks) involved in the Pα2–CL interaction and the cardiolipin headgroup swing (curved arrow).

Extended Data Fig. 7 Examining the electrostatic and hydrophobic distribution of the GDP-AlFx-bound s-OPA1 helical assembly.

The apo state is indicated by dashed boxes. a, A top view of the s-OPA1 GDP-AlFx helical assembly coloured by Coulombic electrostatic potential: positive, blue; negative, red; neutral, white. A dashed black line marking the membrane interface. The flexible helices, Pα2 and Pα6, in the paddle tip and hinge line the membrane interface with positive-charge, but become neutral near where they insert into the lipid tails. b, A clipped view of the helical assembly (top) and a single s-OPA1 dimer coloured by electrostatic potential as in a (inset), focusing on the paddles on the membrane to highlight the positive-charge distribution on the bilayer interface. Pink and blue boxes highlight the helices in the paddle which insert into the membrane, the membrane-inserting loop (Pα2) and Pα6. The Blue boxed region (bottom) shows Pα6 in the context of interface 4. c, A s-OPA1 GDP-AlFx dimer coloured by electrostatic potential and oriented as in a in the side view of the helical assembly. Charge striation is particularly apparent along the dimer within the interlocked stalk interface 1 and at the site of N-terminal coiled-coil interface 8. Zoomed, the striated stalk–stalk interface 1 is highlighted, charges are labelled in green. d, The striated s-OPA1 apo stalk–stalk interface 1 is shown with charges (green) slightly out of register. e, A magnified view of the electrostatic potential for the N-terminal interface 8. f, The inter-rung interface 7 coloured by electrostatic potential, highlighting charge dependence. g–l, Same as in a–f for calculated lipophilicity: hydrophilic, teal; hydrophobic, gold; neutral, white. Hydrophobic regions (gold) within the paddle domain are membrane-inserted (h). Coulombic electrostatic potential and molecular lipophilicity were determined using ChimeraX (ref. 68).

Extended Data Fig. 8 Potential residues involved in s-OPA1 helical interfaces, highlighting the atrophy mutants located within each interface.

a, s-OPA1 GDP-AlFx with all interfacial residues of the helical assembly shown as sticks. The monomer is coloured by domain: N terminus, light grey; GTPase domain, light green; BSE, light pink; hinge 1, light grey; stalks, light blue; paddles, light orange. All atrophy mutants are coloured in red. b, Atrophy mutants located within the buried interfaces. The model is coloured as in a and sticks are shown for interfacial residues with atrophy mutants highlighted in red. Chain A is coloured a dark hue and chain B a light hue. c–h, s-OPA1 interfaces with sticks shown for the buried residues and atrophy mutants coloured in red32. c, s-OPA1 dimer interface between the stalks and paddles (interfaces 1–3). d, Interface 5 between GTPase domains. e, Interface 4 between the paddle hinges. f, The GTPase domain–stalk interface 6 for the A-chains (left) and B-chains (right). g, Interface 7 between inter-rung paddles. h, Interface 8 involving the N-terminal coiled-coil, coloured purple. i, Interface 9, N terminus to BSE interface for B- (left) and A-chains (right) (coloured as in h). In all cases buried interfacial residues were determined using PDBePISA (ref. 32).

Extended Data Fig. 9 Cell images examining the functional effects of all OPA1 interface and membrane-binding mutants.

a–s, Left, mitochondria immunostained against TOM20. Middle, OPA1–GFP localization to the mitochondria. Right, colocalization of OPA1–GFP and mitochondria immunostained against TOM20. Magnified views of the residues in each set of mutations are shown to the left of the corresponding cell images. Monomers coloured as by domain (see Fig. 1). The N-terminal domain is coloured purple. Chain A is coloured a dark hue and chain B a light hue. H-bonding residues are shown in sticks. Residues mutated in the interface are coloured red and labelled. Residues in red associated with DOA mutations are in bold. a, Wild-type OPA1–GFP. b, OPA1–GFP interface 1(a) mutations (R627A:K663A:R683A). c, OPA1–GFP interface 1(b) mutations (E626A:T630A:K668A:E671A). d, OPA1–GFP interface 1(c) mutation (Q659A). e, OPA1–GFP interface 1(d) mutation (E679A). f, OPA1–GFP interface 1(e) mutation (H631A). g, OPA1–GFP interface 2 mutations (K614A:H615A:D835A). h, OPA1–GFP interface 4(a) and membrane-binding Pα6 mutations (F860A:Y861A:Y862A:Y863A:Q864A:R865A:H866A:F867A). i, OPA1–GFP interface 4(b) mutation (I735A). j, OPA1–GFP interface 5 mutation (E444A). k, OPA1–GFP interface 6 mutations (D716A). l, OPA1–GFP interface 7 mutations (K819A:N820A). m, OPA1–GFP interface 8 mutation (R235A). n, OPA1–GFP interface 9 mutations (E213A:Q217A:R557A:D565A). o, OPA1–GFP Membrane-inserting loop, Pα2 mutations (W771A:K772A:K773A:R774A:W775A:L776A:Y777A:W778A:K779A:N780A:R781A). p, OPA1–GFP control 1 non-interfacial mutant (L243A). q, OPA1–GFP control 2 non-interfacial mutant (L248A). r, OPA1–GFP control 3 non-interfacial mutant (K369A). s, OPA1–GFP control 4 non-interfacial mutant (T651A). Scale bars, 10 μm. All experiments were repeated three times; each experiment was repeated independently with similar results.

Extended Data Fig. 10 Cell-imaging statistics examining the functional effects of OPA1 mutants in more detail.

a, Example images of filamentous, intermediate and fragmented mitochondrial networks used for categorizing single-cell data. Left, mitochondria immunostained against TOM20. Middle, OPA1–GFP mitochondrial localization. Right, colocalization of OPA1–GFP and mitochondria immunostained against TOM20. Scale bars, 10 μm. b, Magnified regions of the anti-TOM20 (yellow dash) and merged (pink dash) filamentous, intermediate and fragmented mitochondria. Scale bars, 10 μm. c, Western blot of HeLa-M cells showing the ratios of GFP-tagged and endogenous long- and short-OPA1. Top, long- and short-OPA1–GFP (117 kDa, 129 kDa) detected with an anti-GFP antibody (long- to short-OPA1 ratio: around 0.78:1). The relative expression between WT versus mutant OPA1–GFP is 1:0.8-1.1. Middle, long- and short- ratios of endogenous OPA1 isoforms (~89 kDa, ~101 kDa) detected with anti-OPA1 antibody (long- and short-OPA1 ratio: around 0.8:1). Bottom panel, actin control detected with anti-actin antibody. Below, transfection efficiencies, measured in parallel samples using fluorescence imaging. d, Plot of cells transfected with OPA1–GFP expression levels estimated from differences in intensity relative to background cells. OPA1–GFP mutations are found in Fig. 4 and Extended Data Fig. 9. e, Box and whisker plot of transfected OPA-GFP mutants intensity over background cells. The data are presented as mean ± s.e.m. expression values. The values are 1.59 ±0.12 for cells scored as filamentous, 1.98 ± 0.16 for cells scored as intermediate and 2.04 ± 0.12 for cells scored as fragmented. The numbers are from three biologically independent experiments for all mutants tested. The numbers are from three biologically independent experiments for all mutants tested. The minima are 0.92, 0.98 and 1.32 for filamentous, intermediate and fragmented, respectively. The maxima 2.28, 2.90 and 2.66 for filamentous, intermediate and fragmented, respectively. The medians are 1.51, 1.78 and 2.00 for filamentous, intermediate and fragmented, respectively. The boundaries of the boxes that correspond to 25% and 75% percentiles are 1.24 and 1.95 for filamentous, 1.56 and 2.57 for intermediate and 1.65 and 2.53 for fragmented. f, Box and whisker plot of transfected OPA-GFP WT and controls intensity over background cells. The data are presented as mean ± s.e.m. expression values. The values are 1.89 ± 0.06 for cells scored as filamentous, 1.99 ± 0.25 for cells scored as intermediate and 2.35 ± 0.30 for cells scored as fragmented. The numbers are from three biologically independent experiments for WT and each control. The minima are 1.71, 1.43 and 1.76 for filamentous, intermediate and fragmented, respectively. The maxima are 2.05, 2.90 and 3.18 for filamentous, intermediate and fragmented, respectively. The medians are 1.89, 1.97 and 2.01 for filamentous, intermediate and fragmented, respectively. The boundaries of boxes that correspond to 25% and 75% percentiles are 1.77 and 2.02 for filamentous, 1.52 and 2.46 for intermediate and 1.80 and 3.08 for fragmented. g, Table of t-test values display no significant difference in expression intensity between transfected OPA-GFP mutants and transfected OPA1–GFP WT and control cells. The Student’s t-test (two-sided) was performed using the 2-tailed version, and the two-sample equal variance type. P values of the relative expression levels between WT control versus mutants are 0.140, 0.968 and 0.239 for filamentous, intermediate and fragmented, respectively. They are all not statistically different. Colouring for d–g represents mitochondrial networks categorized as filamentous (blue), intermediate (orange) or fragmented (grey). h–j, FIB-SEM images of mitochondrial cristae from HeLa-M cells transfected with OPA1–GFP. Scale bar, 250 nm. h, Images of OPA1–GFP WT. i, OPA1–GFP I1(a). j, OPA1–GFP Pα2. k–m, TEM images of mitochondrial cristae from HeLa-M cells transfected with OPA1–GFP. Scale bar, 250 nm. k, Images of OPA1–GFP. l, OPA1–GFP I1(a). m, OPA1–GFP Pα2. For a–m, experiments were repeated three times; each experiment was repeated independently with similar results.

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Extended Data Table 1 DOA mutations currently identified in OPA1
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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1–3 and Supplementary Tables 1–3.

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Supplementary Video 1

Morphs between the Models of the Apo and GDP-AlFx-bound state of s-OPA1. Upon transitioning between the Apo to the nucleotide state (GDP-AlFx) the helical assembly compacts due to dimerization of the GTPase domain. In addition, the paddle domain inserts further into the membrane (transparent grey) in the nucleotide state as seen in the cross section and top views of the models.

Supplementary Video 2

Transition of the helical assembly with and without nucleotide from cryoDRGN. The flexibility and heterogeneity of s-OPA1 was examined by cryoDRGN, which revealed continuous transitions in the N-terminal and GTPase-dimer domains as well as in the lipid inserting regions, Pα2 and interface 4.

Supplementary Video 3

Transition from a wide-diameter OPA1 assembly (Zhang et al., 2020) to narrower-diameter apo and nucleotide-bound assemblies. Docking our model into the wider s-OPA1 assembly from Zhang et al., 202022, illustrates how the biological rung elongates as the tube diameter decreases, and slightly adjusts upon nucleotide binding.

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Nyenhuis, S.B., Wu, X., Strub, MP. et al. OPA1 helical structures give perspective to mitochondrial dysfunction. Nature 620, 1109–1116 (2023). https://doi.org/10.1038/s41586-023-06462-1

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