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  • 1.
    Aibara, Shintaro
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Andréll, Juni
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Singh, Vivek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Rapid Isolation of the Mitoribosome from HEK Cells2018In: Journal of Visualized Experiments, E-ISSN 1940-087X, no 140, article id e57877Article in journal (Refereed)
    Abstract [en]

    The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondria! genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart. Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only similar to 10(10) cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

  • 2.
    Aibara, Shintaro
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Singh, Vivek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Modelska, Angelika
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Structural basis of mitochondrial translation2020In: eLIFE, E-ISSN 2050-084X, Vol. 9, article id e58362Article in journal (Refereed)
    Abstract [en]

    Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report similar to 3.0 angstrom resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.

  • 3.
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The revolution evolution2022In: Nature Plants, ISSN 2055-0278, Vol. 8, no 1, p. 14-17Article in journal (Other academic)
  • 4. Bigalke, Janna M.
    et al.
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Roth, Robert
    Dahl, Göran
    Gordon, Euan
    Dorbéus, Sarah
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Sandmark, Jenny
    Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain2019In: Science Advances, E-ISSN 2375-2548, Vol. 5, no 7, article id eaau4202Article in journal (Refereed)
    Abstract [en]

    Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFR alpha 2 and determined its structure at 5.7-angstrom resolution by cryo-EM. The proteins form an assembly through RET-GFR alpha 2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFR alpha 2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.

  • 5. Brown, Alan
    et al.
    Rathore, Sorbhi
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kimanius, Dari
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Bai, Xiao-chen
    Rorbach, Joanna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). MRC Laboratory of Molecular Biology, UK.
    Ramakrishnan, V.
    Structures of the human mitochondrial ribosome in native states of assembly2017In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 24, no 10, p. 866-869Article in journal (Refereed)
    Abstract [en]

    Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to similar to 3-angstrom resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

  • 6. Bugiardini, Enrico
    et al.
    Mitchell, Alice L.
    Dalla Rosa, Ilaria
    Horning-Do, Hue-Tran
    Pitmann, Alan M.
    Poole, Olivia
    Holton, Janice L.
    Shah, Sachit
    Woodward, Cathy
    Hargreaves, Iain
    Quinlivan, Rosaline
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Wiesner, Rudolf J.
    Houlden, Henry
    Holt, Ian J.
    Hanna, Michael G.
    Pitceathly, Robert D. S.
    Spinazzola, Antonella
    MRPS25 mutations impair mitochondrial translation and cause encephalomyopathy2019In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 28, no 16, p. 2711-2719Article in journal (Refereed)
    Abstract [en]

    Mitochondrial disorders are clinically and genetically heterogeneous and are associated with a variety of disease mechanisms. Defects of mitochondrial protein synthesis account for the largest subgroup of disorders manifesting with impaired respiratory chain capacity; yet, only a few have been linked to dysfunction in the protein components of the mitochondrial ribosomes. Here, we report a subject presenting with dyskinetic cerebral palsy and partial agenesis of the corpus callosum, while histochemical and biochemical analyses of skeletal muscle revealed signs of mitochondrial myopathy. Using exome sequencing, we identified a homozygous variant c.215C>T in MRPS25, which encodes for a structural component of the 28S small subunit of the mitochondrial ribosome (mS25). The variant segregated with the disease and substitutes a highly conserved proline residue with leucine (p.P72L) that, based on the high-resolution structure of the 28S ribosome, is predicted to compromise inter-protein contacts and destabilize the small subunit. Concordant with the in silico analysis, patient's fibroblasts showed decreased levels of MRPS25 and other components of the 28S subunit. Moreover, assembled 28S subunits were scarce in the fibroblasts with mutant mS25 leading to impaired mitochondrial translation and decreased levels of multiple respiratory chain subunits. Crucially, these abnormalities were rescued by transgenic expression of wild-type MRPS25 in the mutant fibroblasts. Collectively, our data demonstrate the pathogenicity of the p.P72L variant and identify MRPS25 mutations as a new cause of mitochondrial translation defect.

  • 7.
    Chen, Ming
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Tsinghua University, China.
    Perez-Boerema, Annemarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Zhang, Laixing
    Li, Yanxue
    Yang, Maojun
    Li, Shizhong
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Distinct structural modulation of photosystem I and lipid environment stabilizes its tetrameric assembly2020In: Nature Plants, ISSN 2055-026X, Vol. 6, no 3, p. 314-+Article in journal (Refereed)
    Abstract [en]

    Photosystem I (PSI) is able to form different oligomeric states across various species. To reveal the structural basis for PSI dimerization and tetramerization, we structurally investigated PSI from the cyanobacterium Anabaena. This revealed a disrupted trimerization domain due to lack of the terminal residues of PsaL in the lumen, which resulted in PSI dimers with loose connections between monomers and weaker energy-coupled chlorophylls than in the trimer. At the dimer surface, specific phospholipids, cofactors and interactions in combination facilitated recruitment of another dimer to form a tetramer. Taken together, the relaxed luminal connections and lipid specificity at the dimer interface account for membrane curvature. PSI tetramer assembly appears to increase the surface area of the thylakoid membrane, which would contribute to PSI crowding. Photosystem I from the cyanobacterium Anabaena has a disrupted trimerization domain resulting in dimers with loose connections between monomers. Phospholipids and cofactors at the dimer surface facilitate further dimerization to form a tetramer.

  • 8.
    Cottilli, Patrick
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Itoh, Yuzuru
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nobe, Yuko
    Petrov, Anton S.
    Lisón, Purificación
    Taoka, Masato
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cryo-EM structure and rRNA modification sites of a plant ribosome2022In: Plant Communications, ISSN 2590-3462, Vol. 3, no 5, article id 100342Article in journal (Refereed)
    Abstract [en]

    Protein synthesis in crop plants contributes to the balance of food and fuel on our planet, which influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via the deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and a quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins and 71 plant-specific rRNA modifications, and it re-annotates 30 rRNA residues in the available sequence. At the protein level, isoAsp is found in position 137 of uS11, and a zinc finger previously believed to be universal is missing from eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. At the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation, highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.

  • 9. Desai, Nirupa
    et al.
    Brown, Alan
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). MRC Laboratory of Molecular Biology, UK.
    Ramakrishnan, V.
    The structure of the yeast mitochondrial ribosome2017In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 355, no 6324, p. 528-531Article in journal (Refereed)
    Abstract [en]

    Mitochondria have specialized ribosomes (mitoribosomes) dedicated to the expression of the genetic information encoded by their genomes. Here, using electron cryomicroscopy, we have determined the structure of the 75-component yeast mitoribosome to an overall resolution of 3.3 angstroms. The mitoribosomal small subunit has been built de novo and includes 15S ribosomal RNA (rRNA) and 34 proteins, including 14 without homologs in the evolutionarily related bacterial ribosome. Yeast-specific rRNA and protein elements, including the acquisition of a putatively active enzyme, give the mitoribosome a distinct architecture compared to the mammalian mitoribosome. At an expanded messenger RNA channel exit, there is a binding platform for translational activators that regulate translation in yeast but not mammalian mitochondria. The structure provides insights into the evolution and species-specific specialization of mitochondrial translation.

  • 10.
    Forsberg, Björn O.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kimanius, Dari
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Paul, Bijoya
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Cryo-EM reconstruction of the chlororibosome to 3.2 angstrom resolution within 24 h2017In: IUCrJ, E-ISSN 2052-2525, Vol. 4, p. 723-727Article in journal (Refereed)
    Abstract [en]

    The introduction of direct detectors and the automation of data collection in cryo-EM have led to a surge in data, creating new opportunities for advancing computational processing. In particular, on-the-fly workflows that connect data collection with three-dimensional reconstruction would be valuable for more efficient use of cryo-EM and its application as a sample-screening tool. Here, accelerated on-the-fly analysis is reported with optimized organization of the data-processing tools, image acquisition and particle alignment that make it possible to reconstruct the three-dimensional density of the 70S chlororibosome to 3.2 angstrom resolution within 24 h of tissue harvesting. It is also shown that it is possible to achieve even faster processing at comparable quality by imposing some limits to data use, as illustrated by a 3.7 angstrom resolution map that was obtained in only 80 min on a desktop computer. These on-the-fly methods can be employed as an assessment of data quality from small samples and extended to high-throughput approaches.

  • 11. Gahura, Ondřej
    et al.
    Mühleip, Alexander
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hierro-Yap, Carolina
    Panicucci, Brian
    Jain, Minal
    Hollaus, David
    Slapničková, Martina
    Zíková, Alena
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, no 1, article id 5989Article in journal (Refereed)
    Abstract [en]

    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. Mitochondrial ATP synthase assemble into oligomers. Here, authors resolve the structure of trypanosomal ATP synthase, showing that its dimerization is essential for function and evolutionary conserved.

  • 12.
    Itoh, Yuzuru
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Andréll, Juni
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Choi, Austin
    Richter, Uwe
    Maiti, Priyanka
    Best, Robert B.
    Barrientos, Antoni
    Battersby, Brendan J.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Mechanism of membrane-tethered mitochondrial protein synthesis2021In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 371, no 6531, p. 846-849Article in journal (Refereed)
    Abstract [en]

    Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.

  • 13.
    Itoh, Yuzuru
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Khawaja, Anas
    Laptev, Ivan
    Cipullo, Miriam
    Atanassov, Ilian
    Sergiev, Petr
    Rorbach, Joanna
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mechanism of mitoribosomal small subunit biogenesis and preinitiation2022In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 606, p. 603-608Article in journal (Refereed)
    Abstract [en]

    Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small mitoribosomal subunit (SSU) intermediates in complex with auxiliary factors, revealing a sequential assembly mechanism. The methyltransferase TFB1M binds to partially unfolded rRNA h45 that is promoted by RBFA, while the mRNA channel is blocked. This enables binding of METTL15 that promotes further rRNA maturation and a large conformational change of RBFA. The new conformation allows initiation factor mtIF3 to already occupy the subunit interface during the assembly. Finally, the mitochondria-specific ribosomal protein mS37 (ref. 1) outcompetes RBFA to complete the assembly with the SSU–mS37–mtIF3 complex2 that proceeds towards mtIF2 binding and translation initiation. Our results explain how the action of step-specific factors modulate the dynamic assembly of the SSU, and adaptation of a unique protein, mS37, links the assembly to initiation to establish the catalytic human mitoribosome.

  • 14.
    Itoh, Yuzuru
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Naschberger, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mortezaei, Narges
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Herrmann, Johannes M.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Analysis of translating mitoribosome reveals functional characteristics of translation in mitochondria of fungi2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 5187Article in journal (Refereed)
    Abstract [en]

    Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.

  • 15.
    Itoh, Yuzuru
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Singh, Vivek
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Khawaja, Anas
    Naschberger, Andreas
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nguyen, Minh Duc
    Rorbach, Joanna
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules2022In: eLIFE, E-ISSN 2050-084X, Vol. 11, article id e77460Article in journal (Refereed)
    Abstract [en]

    The mitoribosome regulates cellular energy production, and its dysfunction is associated with aging. Inhibition of the mitoribosome can be caused by off-target binding of antimicrobial drugs and was shown to be coupled with a bilateral decreased visual acuity. Previously, we reported mitochondria-specific protein aspects of the mitoribosome, and in this article we present a 2.4-Å resolution structure of the small subunit in a complex with the anti-tuberculosis drug streptomycin that reveals roles of non-protein components. We found iron–sulfur clusters that are coordinated by different mitoribosomal proteins, nicotinamide adenine dinucleotide (NAD) associated with rRNA insertion, and posttranslational modifications. This is the first evidence of inter-protein coordination of iron–sulfur, and the finding of iron–sulfur clusters and NAD as fundamental building blocks of the mitoribosome directly links to mitochondrial disease and aging. We also report details of streptomycin interactions, suggesting that the mitoribosome-bound streptomycin is likely to be in hydrated gem-diol form and can be subjected to other modifications by the cellular milieu. The presented approach of adding antibiotics to cultured cells can be used to define their native structures in a bound form under more physiological conditions, and since streptomycin is a widely used drug for treatment, the newly resolved features can serve as determinants for targeting.

  • 16. Khawaja, Anas
    et al.
    Itoh, Yuzuru
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Remes, Cristina
    Spåhr, Henrik
    Yukhnovets, Olessya
    Höfig, Henning
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Rorbach, Joanna
    Distinct pre-initiation steps in human mitochondrial translation2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 2932Article in journal (Refereed)
    Abstract [en]

    Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.

  • 17.
    Kock Flygaard, Rasmus
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institute, Sweden.
    Mühleip, Alexander
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institute, Sweden.
    Tobiasson, Victor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institute, Sweden.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institute, Sweden.
    Type III ATP synthase is a symmetry-deviated dimer that induces membrane curvature through tetramerization2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 5342Article in journal (Refereed)
    Abstract [en]

    Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF1. Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit-a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 angstrom resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature.

  • 18. Matzov, Donna
    et al.
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Basu, Arnab
    Zimmerman, Ella
    Bashan, Anat
    Yap, Mee-Ngan F.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Yonath, Ada E.
    The cryo-EM structure of hibernating 100S ribosome dimer from pathogenic Staphylococcus aureus2017In: Nature Communications, E-ISSN 2041-1723, Vol. 8, article id 723Article in journal (Refereed)
    Abstract [en]

    Formation of 100S ribosome dimer is generally associated with translation suppression in bacteria. Trans-acting factors ribosome modulation factor (RMF) and hibernating promoting factor (HPF) were shown to directly mediate this process in E. coli. Gram-positive S. aureus lacks an RMF homolog and the structural basis for its 100S formation was not known. Here we report the cryo-electron microscopy structure of the native 100S ribosome from S. aureus, revealing the molecular mechanism of its formation. The structure is distinct from previously reported analogs and relies on the HPF C-terminal extension forming the binding platform for the interactions between both of the small ribosomal subunits. The 100S dimer is formed through interactions between rRNA h26, h40, and protein uS2, involving conformational changes of the head as well as surface regions that could potentially prevent RNA polymerase from docking to the ribosome.

  • 19.
    Mühleip, Alexander
    et al.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institute, Sweden.
    Flygaard, Rasmus Kock
    Ovciarikova, Jana
    Lacombe, Alice
    Fernandes, Paula
    Sheiner, Lilach
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institute, Sweden.
    ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria2021In: Nature Communications, E-ISSN 2041-1723, Vol. 12, no 1, article id 120Article in journal (Refereed)
    Abstract [en]

    Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.

  • 20.
    Mühleip, Alexander
    et al.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kock Flygaard, Rasmus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Baradaran, Rozbeh
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haapanen, Outi
    Gruhl, Thomas
    Tobiasson, Victor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Maréchal, Amandine
    Sharma, Vivek
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural basis of mitochondrial membrane bending by the I–II–III2–IV2 supercomplex2023In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 615, no 7954, p. 934-938Article in journal (Refereed)
    Abstract [en]

    Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I–II–III2–IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization

  • 21.
    Mühleip, Alexander
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    McComas, Sarah E.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Structure of a mitochondrial ATP synthase with bound native cardiolipin2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e51179Article in journal (Refereed)
    Abstract [en]

    The mitochondrial ATP synthase fuels eukaryotic cells with chemical energy. Here we report the cryo-EM structure of a divergent ATP synthase dimer from mitochondria of Euglena gracilis, a member of the phylum Euglenozoa that also includes human parasites. It features 29 different subunits, 8 of which are newly identified. The membrane region was determined to 2.8 angstrom resolution, enabling the identification of 37 associated lipids, including 25 cardiolipins, which provides insight into protein-lipid interactions and their functional roles. The rotor-stator interface comprises four membrane-embedded horizontal helices, including a distinct subunit a. The dimer interface is formed entirely by phylum-specific components, and a peripherally associated subcomplex contributes to the membrane curvature. The central and peripheral stalks directly interact with each other. Last, the ATPase inhibitory factor 1 (IF1) binds in a mode that is different from human, but conserved in Trypanosomatids.

  • 22.
    Naschberger, Andreas
    et al.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fadeeva, Mariia
    Klaiman, Daniel
    Borovikova-Sheinker, Anna
    Caspy, Ido
    Nelson, Nathan
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Westlake University, China.
    Structure of plant photosystem I in a native assembly state defines PsaF as a regulatory checkpoint2024In: Nature plants, ISSN 2055-026XArticle in journal (Refereed)
    Abstract [en]

    Plant photosystem I (PSI) consists of at least 13 nuclear-encoded and 4 chloroplast-encoded subunits that together act as a sunlight-driven oxidoreductase. Here we report the structure of a PSI assembly intermediate that we isolated from greening oat seedlings. The assembly intermediate shows an absence of at least eight subunits, including PsaF and LHCI, and lacks photoreduction activity. The data show that PsaF is a regulatory checkpoint that promotes the assembly of LHCI, effectively coupling biogenesis to function. This study reports the structure of a photosystem I assembly intermediate isolated from greening oat seedlings. It defines PsaF as a regulatory checkpoint promoting the association of LHCI that couples biogenesis to function.

  • 23.
    Naschberger, Andreas
    et al.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mosebach, Laura
    Tobiasson, Victor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kuhlgert, Sebastian
    Scholz, Martin
    Perez Boerema, Annemarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ho, Thi Thu Hoai
    Vidal-Meireles, André
    Takahashi, Yuichiro
    Hippler, Michael
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Algal photosystem I dimer and high-resolution model of PSI-plastocyanin complex2022In: Nature Plants, ISSN 2055-0278, Vol. 8, no 10, p. 1191-1201Article in journal (Refereed)
    Abstract [en]

    Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI–light-harvesting complex I monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH and thus partially overlaps with the surface area that would bind one of the light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI–light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways.

  • 24. Nirwan, Neha
    et al.
    Itoh, Yuzuru
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Singh, Pratima
    Bandyopadhyay, Sutirtha
    Vinothkuman, Kutti R.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Saikrishnan, Kayarat
    Structure-based mechanism for activation of the AAA plus GTPase McrB by the endonuclease McrC2019In: Nature Communications, E-ISSN 2041-1723, Vol. 10, article id 3058Article in journal (Refereed)
    Abstract [en]

    The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 angstrom structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the gamma subunit complexed to alpha(3)beta(3) of F-1-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5'-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.

  • 25.
    Ott, Martin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Brown, Alan
    Organization and Regulation of Mitochondrial Protein Synthesis2016In: Annual Review of Biochemistry, ISSN 0066-4154, E-ISSN 1545-4509, Vol. 85, p. 77-101Article, review/survey (Refereed)
    Abstract [en]

    Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria.

  • 26.
    Perez Boerema, Annemarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Paul, Bijoya
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Tobiasson, Victor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kimanius, Dari
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Forsberg, Björn O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Walldén, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor2018In: Nature Plants, ISSN 2055-026X, Vol. 4, p. 212-217Article in journal (Refereed)
    Abstract [en]

    Oxygenic photosynthesis produces oxygen and builds a variety of organic compounds, changing the chemistry of the air, the sea and fuelling the food chain on our planet. The photochemical reactions underpinning this process in plants take place in the chloroplast. Chloroplasts evolved ~1.2 billion years ago from an engulfed primordial diazotrophic cyanobacterium, and chlororibosomes are responsible for synthesis of the core proteins driving photochemical reactions. Chlororibosomal activity is spatiotemporally coupled to the synthesis and incorporation of functionally essential co-factors, implying the presence of chloroplast-specific regulatory mechanisms and structural adaptation of the chlororibosome1,2. Despite recent structural information3,4,5,6, some of these aspects remained elusive. To provide new insights into the structural specialities and evolution, we report a comprehensive analysis of the 2.9–3.1 Å resolution electron cryo-microscopy structure of the spinach chlororibosome in complex with its recycling factor and hibernation-promoting factor. The model reveals a prominent channel extending from the exit tunnel to the chlororibosome exterior, structural re-arrangements that lead to increased surface area for translocon binding, and experimental evidence for parallel and convergent evolution of chloro- and mitoribosomes.

  • 27.
    Perez-Boerema, Annemarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Klaiman, Daniel
    Caspy, Ido
    Netzer-El, Sigal Y.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Nelson, Nathan
    Structure of a minimal photosystem I from the green alga Dunaliella salina2020In: Nature plants, ISSN 2055-026X, Vol. 6, no 3, p. 321-327Article in journal (Refereed)
    Abstract [en]

    Solar energy harnessed by oxygenic photosynthesis supports most of the life forms on Earth. In eukaryotes, photosynthesis occurs in chloroplasts and is achieved by membrane-embedded macromolecular complexes that contain core and peripheral antennae with multiple pigments. The structure of photosystem I (PSI) comprises the core and light-harvesting (LHCI) complexes, which together form PSI-LHCI. Here we determined the structure of PSI-LHCI from the salt-tolerant green alga Dunaliella salina using X-ray crystallography and electron cryo-microscopy. Our results reveal a previously undescribed configuration of the PSI core. It is composed of only 7 subunits, compared with 14-16 subunits in plants and the alga Chlamydomonas reinhardtii, and forms the smallest known PSI. The LHCI is poorly conserved at the sequence level and binds to pigments that form new energy pathways, and the interactions between the individual Lhca1-4 proteins are weakened. Overall, the data indicate the PSI of D. salina represents a different type of the molecular organization that provides important information for reconstructing the plasticity and evolution of PSI. The photosystem I light-harvesting complex from the salt-tolerant green alga Dunaliella salina has a core configuration composed of only seven subunits. This unusual molecular organization could inform the reconstruction of photosystem evolution.

  • 28. Petrov, Anton S.
    et al.
    Wood, Elizabeth C.
    Bernier, Chad R.
    Norris, Ashlyn M.
    Brown, Alan
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Structural Patching Fosters Divergence of Mitochondrial Ribosomes2019In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 36, no 2, p. 207-219Article in journal (Refereed)
    Abstract [en]

    Mitochondrial ribosomes (mitoribosomes) are essential components of all mitochondria that synthesize proteins encoded by the mitochondrial genome. Unlike other ribosomes, mitoribosomes are highly variable across species. The basis for this diversity is not known. Here, we examine the composition and evolutionary history of mitoribosomes across the phylogenetic tree by combining three-dimensional structural information with a comparative analysis of the secondary structures of mitochondrial rRNAs (mt-rRNAs) and available proteomic data. We generate a map of the acquisition of structural variation and reconstruct the fundamental stages that shaped the evolution of the mitoribosomal large subunit and led to this diversity. Our analysis suggests a critical role for ablation and expansion of rapidly evolving mt-rRNA. These changes cause structural instabilities that are patched by the acquisition of pre-existing compensatory elements, thus providing opportunities for rapid evolution. This mechanism underlies the incorporation of mt-tRNA into the central protuberance of the mammalian mitoribosome, and the altered path of the polypeptide exit tunnel of the yeast mitoribosome. We propose that since the toolkits of elements utilized for structural patching differ between mitochondria of different species, it fosters the growing divergence of mitoribosomes.

  • 29. Sighel, Denise
    et al.
    Notarangelo, Michela
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Re, Angela
    Ricci, Gianluca
    Guida, Marianna
    Soldano, Alessia
    Adami, Valentina
    Ambrosini, Chiara
    Broso, Francesca
    Rosatti, Emanuele Filiberto
    Longhi, Sara
    Buccarelli, Mariachiara
    D'Alessandris, Quintino G.
    Giannetti, Stefano
    Pacioni, Simone
    Ricci-Vitiani, Lucia
    Rorbach, Joanna
    Pallini, Roberto
    Roulland, Sandrine
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mancini, Ines
    Modelska, Angelika
    Quattrone, Alessandro
    Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth2021In: Cell Reports, E-ISSN 2211-1247, Vol. 35, no 4, article id 109024Article in journal (Refereed)
    Abstract [en]

    Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.

  • 30.
    Singh, Vivek
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Conor Moran, J.
    Itoh, Yuzuru
    Soto, Iliana C.
    Fontanesi, Flavia
    Couvillion, Mary
    Huynen, Martijn A.
    Churchman, Stirling
    Barrientos, Antoni
    Amunts, Alexey
    Structural basis of LRPPRC-SLIRP-dependent translation by the mitoribosome2022Manuscript (preprint) (Other academic)
  • 31.
    Tobiasson, Victor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation2020In: eLIFE, E-ISSN 2050-084X, Vol. 9, article id e59264Article in journal (Refereed)
    Abstract [en]

    To understand the steps involved in the evolution of translation, we used Tetrahymena thermophila, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal proteins.

  • 32.
    Tobiasson, Victor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure of a mitochondrial ribosome with fragmented rRNA in complexwith membrane-targeting elementsManuscript (preprint) (Other academic)
    Abstract [en]

    Mitoibosomes of green alga belong to a diverged type with extreme fragmentation in rRNA andprotein material. We present a 2.9-Å resolution structure of an algal mitoribosome with reducedrRNA that is split in to 13 fragments. The architecture features a fragmentation pattern that hassimilarities with apicomplexan parasites. The unique phenomena of the rRNA include permutationand incorporation of a non-canonical and reduced mt-5S rRNA. On the protein level, elevenperipherally associated HEAT-repeat proteins involved in rRNA binding, and a specific trimer ofmL116 binds 3’ termini of three rRNA fragments. In the exit tunnel, mL128 constricts the path,and mL105 a homolog of a membrane targeting component mediates contacts with an innermembrane-bound insertase. Although overall protein content at the tunnel exit site of themitoribosome varies among eukaryotes, we find that the mechanisms of the constriction andmembrane tethering are shared between the algal and mammalian lineages. Therefore, our datareveals characteristics of fragmented rRNA an unexpected convergent evolution in the regulationof protein synthesis in mitochondria.

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  • 33.
    Tobiasson, Victor
    et al.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berzina, Ieva
    Amunts, Alexey
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure of a mitochondrial ribosome with fragmented rRNA in complex with membrane-targeting elements2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, no 1, article id 6132Article in journal (Refereed)
    Abstract [en]

    Mitoribosomes of green algae display a great structural divergence from their tracheophyte relatives, with fragmentation of both rRNA and proteins as a defining feature. Here, we report a 2.9 angstrom resolution structure of the mitoribosome from the alga Polytomella magna harbouring a reduced rRNA split into 13 fragments. We found that the rRNA contains a non-canonical reduced form of the 5S, as well as a permutation of the LSU domain I. The mt-5S rRNA is stabilised by mL40 that is also found in mitoribosomes lacking the 5S, which suggests an evolutionary pathway. Through comparison to other ribosomes with fragmented rRNAs, we observe that the pattern is shared across large evolutionary distances, and between cellular compartments, indicating an evolutionary convergence and supporting the concept of a primordial fragmented ribosome. On the protein level, eleven peripherally associated HEAT-repeat proteins are involved in the binding of 3' rRNA termini, and the structure features a prominent pseudo-trimer of one of them (mL116). Finally, in the exit tunnel, mL128 constricts the tunnel width of the vestibular area, and mL105, a homolog of a membrane targeting component mediates contacts with an inner membrane bound insertase. Together, the structural analysis provides insight into the evolution of the ribosomal machinery in mitochondria.

  • 34.
    Tobiasson, Victor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Dow, Allexa
    Prisic, Sladjana
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Zinc depletion does not necessarily induce ribosome hibernation in mycobacteria2019In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 7, p. 2395-2397Article in journal (Refereed)
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  • 35.
    Tobiasson, Victor
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Gahura, Ondřej
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Baradaran, Rozbeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ziková, Alena
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit2021In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 40, no 6, article id e106292Article in journal (Refereed)
    Abstract [en]

    Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 angstrom, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

  • 36.
    Wú, Fēi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mühleip, Alexander
    Gruhl, Thomas
    Sheiner, Lilach
    Maréchal, Amandine
    Amunts, Alexey
    Structure of the II2-III2-IV2 mitochondrial supercomplex from the parasite Perkinsus marinus2024Manuscript (preprint) (Other academic)
    Abstract [en]

    Respiratory complexes have co-evolved into supercomplexes in different clades to sustain energy production at the basis of eukaryotic life. In this study, using cryogenic electron microscopy, we determined the 2.1 Å resolution structure of a 104-subunit II2-III2-IV2 supercomplex from the parasite Perkinsus marinus, related to Apicomplexa, capable of complete electron transport from succinate to molecular oxygen. A feature of the parasite is the association of two copies of complex II via the apicomplexan subunit SDHG that interacts with both complexes III and IV and bridge the supercomplex. In the c1 state, we identified two protein factors, ISPR1 and ISPR2 bound on the surface of complex III, where Cytochrome c docks, acting as negative regulators. The acquisition of 15 specific subunits to complex IV results in its lateral offset, increasing the distance between the Cytochrome c electron donor and acceptor sites. The domain homologous to canonical mitochondria-encoded transmembrane subunit COX2 is made of three separate polypeptides encoded in the nucleus, and their correct assembly is a prerequisite for electron transport in the supercomplex. Subunits Cytochrome b and COX1 comprise a +2 frameshift introduced during protein synthesis by the mitoribosome. Among 114 modelled endogenous lipids, we detect a direct contribution to the formation of the divergent supercomplex and its functional sites, including assembly of CII and ubiquinone binding. Together, our findings expose the uniqueness of the principal components of bioenergetics in the mitochondria of parasites.

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