Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

Faculty of Science University of South Bohemia 37005 České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences 37005 České Budějovice Czech Republic

Science for Life Laboratory Department of Biochemistry and Biophysics Stockholm University 17165 Solna Sweden

Zobrazit více v PubMed

Paumard P, et al. The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J. 2002;21:221–230. doi: 10.1093/emboj/21.3.221. PubMed DOI PMC

Davies KM, Anselmi C, Wittig I, Faraldo-Gomez JD, Kuhlbrandt W. Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae. Proc. Natl Acad. Sci. USA. 2012;109:13602–13607. doi: 10.1073/pnas.1204593109. PubMed DOI PMC

Panek T, Elias M, Vancova M, Lukes J, Hashimi H. Returning to the fold for lessons in mitochondrial crista diversity and evolution. Curr. Biol. 2020;30:R575–R588. doi: 10.1016/j.cub.2020.02.053. PubMed DOI

Kuhlbrandt W. Structure and mechanisms of F-Type ATP synthases. Annu. Rev. Biochem. 2019;88:515–549. doi: 10.1146/annurev-biochem-013118-110903. PubMed DOI

Spikes TE, Montgomery MG, Walker JE. Structure of the dimeric ATP synthase from bovine mitochondria. Proc. Natl Acad. Sci. USA. 2020;117:23519–23526. doi: 10.1073/pnas.2013998117. PubMed DOI PMC

Pinke G, Zhou L, Sazanov LA. Cryo-EM structure of the entire mammalian F-type ATP synthase. Nat. Struct. Mol. Biol. 2020;27:1077–1085. doi: 10.1038/s41594-020-0503-8. PubMed DOI

Guo H, Bueler SA, Rubinstein JL. Atomic model for the dimeric Fo region of mitochondrial ATP synthase. Science. 2017;358:936–940. doi: 10.1126/science.aao4815. PubMed DOI PMC

Murphy, B. J. et al. Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling. Science364, eaaw9128 (2019). PubMed

Flygaard RK, Mühleip A, Tobiasson V, Amunts A. Type III ATP synthase is a symmetry-deviated dimer that induces membrane curvature through tetramerization. Nat. Commun. 2020;11:5342. doi: 10.1038/s41467-020-18993-6. PubMed DOI PMC

Muhleip A, McComas SE, Amunts A. Structure of a mitochondrial ATP synthase with bound native cardiolipin. Elife. 2019;8:e51179. doi: 10.7554/eLife.51179. PubMed DOI PMC

Mühleip A, et al. ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria. Nat. Commun. 2021;12:120. doi: 10.1038/s41467-020-20381-z. PubMed DOI PMC

Gahura O, et al. The F1-ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit. FEBS J. 2018;285:614–628. doi: 10.1111/febs.14364. PubMed DOI

Montgomery MG, Gahura O, Leslie AGW, Zikova A, Walker JE. ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites. Proc. Natl Acad. Sci. USA. 2018;115:2102–2107. doi: 10.1073/pnas.1720940115. PubMed DOI PMC

Serricchio M, et al. Depletion of cardiolipin induces major changes in energy metabolism in Trypanosoma brucei bloodstream forms. FASEB J. 2020;35:21176. PubMed

Muhleip AW, Dewar CE, Schnaufer A, Kuhlbrandt W, Davies KM. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits. Proc. Natl Acad. Sci. USA. 2017;114:992–997. doi: 10.1073/pnas.1612386114. PubMed DOI PMC

Schnaufer A, Clark-Walker GD, Steinberg AG, Stuart K. The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function. EMBO J. 2005;24:4029–4040. doi: 10.1038/sj.emboj.7600862. PubMed DOI PMC

Brown SV, Hosking P, Li J, Williams N. ATP synthase is responsible for maintaining mitochondrial membrane potential in bloodstream form Trypanosoma brucei. Eukaryot. Cell. 2006;5:45–53. doi: 10.1128/EC.5.1.45-53.2006. PubMed DOI PMC

Gahura O, Hierro-Yap C, Zikova A. Redesigned and reversed: Architectural and functional oddities of the trypanosomal ATP synthase. Parasitology. 2021;148:1151–1160. doi: 10.1017/S0031182021000202. PubMed DOI PMC

Hierro-Yap C, et al. Bioenergetic consequences of FoF1-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei. J. Biol. Chem. 2021;296:100357. doi: 10.1016/j.jbc.2021.100357. PubMed DOI PMC

Gahura O, Panicucci B, Vachova H, Walker JE, Zikova A. Inhibition of F1-ATPase from Trypanosoma brucei by its regulatory protein inhibitor TbIF1. FEBS J. 2018;285:4413–4423. doi: 10.1111/febs.14672. PubMed DOI

Zikova A, Schnaufer A, Dalley RA, Panigrahi AK, Stuart KD. The F(0)F(1)-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei. PLoS Pathog. 2009;5:e1000436. doi: 10.1371/journal.ppat.1000436. PubMed DOI PMC

Perez E, et al. The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae. Mitochondrion. 2014;19:338–349. doi: 10.1016/j.mito.2014.02.001. PubMed DOI

Sathish Yadav KN, et al. Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis. Biochim. Biophys. Acta. 2017;1858:267–275. doi: 10.1016/j.bbabio.2017.01.007. PubMed DOI

Dewar, C.E., Oeljeklaus, S., Wenger, C., Warscheid, B. & Schneider, A. Characterisation of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei. J. Biol. Chem. 298, 101829 (2022) PubMed PMC

Aphasizheva I, et al. Lexis and grammar of mitochondrial RNA processing in trypanosomes. Trends Parasitol. 2020;36:337–355. doi: 10.1016/j.pt.2020.01.006. PubMed DOI PMC

Blum B, Bakalara N, Simpson L. A model for RNA editing in kinetoplastid mitochondria: “guide” RNA molecules transcribed from maxicircle DNA provide the edited information. Cell. 1990;60:189–198. doi: 10.1016/0092-8674(90)90735-W. PubMed DOI

Adler BK, Harris ME, Bertrand KI, Hajduk SL. Modification of Trypanosoma brucei mitochondrial rRNA by posttranscriptional 3’ polyuridine tail formation. Mol. Cell Biol. 1991;11:5878–5884. PubMed PMC

Hofer A, Steverding D, Chabes A, Brun R, Thelander L. Trypanosoma brucei CTP synthetase: A target for the treatment of African sleeping sickness. Proc. Natl Acad. Sci. USA. 2001;98:6412–6416. doi: 10.1073/pnas.111139498. PubMed DOI PMC

Sobti M, et al. Cryo-EM structures provide insight into how E. coli F1Fo ATP synthase accommodates symmetry mismatch. Nat. Commun. 2020;11:2615. doi: 10.1038/s41467-020-16387-2. PubMed DOI PMC

Gupta K, et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature. 2017;541:421–424. doi: 10.1038/nature20820. PubMed DOI PMC

Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schagger H. Yeast mitochondrial F1Fo-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J. 1998;17:7170–7178. doi: 10.1093/emboj/17.24.7170. PubMed DOI PMC

Gu J, et al. Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1. Science. 2019;364:1068–1075. doi: 10.1126/science.aaw4852. PubMed DOI

Spikes TE, Montgomery MG, Walker JE. Interface mobility between monomers in dimeric bovine ATP synthase participates in the ultrastructure of inner mitochondrial membranes. Proc. Natl Acad. Sci. USA. 2021;118:e2021012118. doi: 10.1073/pnas.2021012118. PubMed DOI PMC

Cadena LR, et al. Mitochondrial contact site and cristae organization system and F1Fo-ATP synthase crosstalk is a fundamental property of mitochondrial cristae. mSphere. 2021;6:e0032721. doi: 10.1128/mSphere.00327-21. PubMed DOI PMC

Davies KM, et al. Macromolecular organization of ATP synthase and complex I in whole mitochondria. Proc. Natl Acad. Sci. USA. 2011;108:14121–14126. doi: 10.1073/pnas.1103621108. PubMed DOI PMC

Blum TB, Hahn A, Meier T, Davies KM, Kühlbrandt W. Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows. Proc. Natl Acad. Sci. USA. 2019;116:4250–4255. doi: 10.1073/pnas.1816556116. PubMed DOI PMC

Bochud-Allemann N, Schneider A. Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei. J. Biol. Chem. 2002;277:32849–32854. doi: 10.1074/jbc.M205776200. PubMed DOI

Poon SK, Peacock L, Gibson W, Gull K, Kelly S. A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor. Open Biol. 2012;2:110037. doi: 10.1098/rsob.110037. PubMed DOI PMC

Allemann N, Schneider A. ATP production in isolated mitochondria of procyclic Trypanosoma brucei. Mol. Biochem. Parasitol. 2000;111:87–94. doi: 10.1016/S0166-6851(00)00303-0. PubMed DOI

Aibara, S., Dienemann, C., & Cramer, P. Structure of an inactive RNA polymerase II dimer. Nucleic Acids Res.49, gkab783 (2021). PubMed PMC

de la Rosa-Trevin JM, et al. Scipion: A software framework toward integration, reproducibility, and validation in 3D electron microscopy. J. Struct. Biol. 2016;195:93–99. doi: 10.1016/j.jsb.2016.04.010. PubMed DOI

Zhang K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 2016;193:1–12. doi: 10.1016/j.jsb.2015.11.003. PubMed DOI PMC

Cowtan K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D. Biol. Crystallogr. 2006;62:1002–1011. doi: 10.1107/S0907444906022116. PubMed DOI

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

Waterhouse A, et al. SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46:W296–W303. doi: 10.1093/nar/gky427. PubMed DOI PMC

Williams CJ, et al. MolProbity: More and better reference data for improved all‐atom structure validation. Protein Sci. 2018;27:293–315. doi: 10.1002/pro.3330. PubMed DOI PMC

Barad BA, et al. EMRinger: Side chain–directed model and map validation for 3D cryo-electron microscopy. Nat. Methods. 2015;12:943–946. doi: 10.1038/nmeth.3541. PubMed DOI PMC

Goddard TD, et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 2018;27:14–25. doi: 10.1002/pro.3235. PubMed DOI PMC

Ho BK, Gruswitz F. HOLLOW: Generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 2008;8:49. doi: 10.1186/1472-6807-8-49. PubMed DOI PMC

Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes

The Ancestral Shape of the Access Proton Path of Mitochondrial ATP Synthases Revealed by a Split Subunit-a