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Singh, V. (2023). Structural investigation of human mitochondrial translation and off-target antibiotic binding. (Doctoral dissertation). Stockholm: Department of Biochemistry and Biophysics, Stockholm University
Open this publication in new window or tab >>Structural investigation of human mitochondrial translation and off-target antibiotic binding
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human mitochondrial translation machinery has evolved to translate 13 mitochondrial mRNAs encoding components of the oxidative phosphorylation pathway responsible for ATP production. The structural basis of human mitochondrial translation is distinct from the canonical bacterial and cytosolic translation systems. Further, mutations affecting mitochondrial protein synthesis disrupt ATP production resulting in myopathies and neurodegenerative diseases. Structural studies have identified the core components of the human mitoribosome and some of its associated translation factors but several important aspects such as the role of mito-specific proteins in translation, rRNA modifications, composition of its ultrastructure including ions, small molecule co-factors, and solvent content, remain poorly understood. Importantly, several important antibiotics that target bacterial translation also affect mitochondrial translation, thereby causing adverse effects in patients. Understanding the mechanism of off-target antibiotic binding to the mitoribosome could help in designing better antibiotics. In this work, we use electron cryo-microscopy to determine the structures of the human mitoribosome in complex with ligands: mRNA/tRNA and translation activators such as LRPPRC-SLIRP. This allows us to explore the structural basis of mitochondrial translation, identifying the roles of mito-specific protein elements in tRNA and mRNA binding and recruitment (Papers 1 and 2). We determine a 2.2 Å resolution structure of the human mitoribosome and a 2.4 Å resolution structure of the mitoribosomal small subunit in complex with the tuberculosis drug, streptomycin. Together, the structures represent the most detailed and complete models for the human mitoribosome, revealing rRNA and protein modifications; several novel small molecule cofactors: 2Fe-2S clusters, polyamines and nucleotides and mechanisms of antibiotic binding (Papers 3 and 4).

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2023. p. 65
Keywords
Mitochondrial translation, mitoribosome, electron cryo-microscopy, LRPPRC-SLIRP, antibiotics, streptomycin
National Category
Structural Biology Biophysics Biochemistry and Molecular Biology
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-215504 (URN)978-91-8014-240-3 (ISBN)978-91-8014-241-0 (ISBN)
Public defence
2023-04-24, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2023-03-30 Created: 2023-03-16 Last updated: 2023-03-24Bibliographically approved
Itoh, Y., Singh, V., Khawaja, A., Naschberger, A., Nguyen, M. D., Rorbach, J. & Amunts, A. (2022). Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules. eLIFE, 11, Article ID e77460.
Open this publication in new window or tab >>Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules
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2022 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 11, article id e77460Article in journal (Refereed) Published
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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-213372 (URN)10.7554/eLife.77460 (DOI)000895763900001 ()36480258 (PubMedID)2-s2.0-85143564523 (Scopus ID)
Available from: 2023-01-09 Created: 2023-01-09 Last updated: 2023-03-16Bibliographically approved
Aibara, S., Singh, V., Modelska, A. & Amunts, A. (2020). Structural basis of mitochondrial translation. eLIFE, 9, Article ID e58362.
Open this publication in new window or tab >>Structural basis of mitochondrial translation
2020 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 9, article id e58362Article in journal (Refereed) Published
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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-185337 (URN)10.7554/eLife.58362 (DOI)000563031000001 ()32812867 (PubMedID)
Available from: 2020-12-03 Created: 2020-12-03 Last updated: 2023-03-16Bibliographically approved
Aibara, S., Andréll, J., Singh, V. & Amunts, A. (2018). Rapid Isolation of the Mitoribosome from HEK Cells. Journal of Visualized Experiments (140), Article ID e57877.
Open this publication in new window or tab >>Rapid Isolation of the Mitoribosome from HEK Cells
2018 (English)In: Journal of Visualized Experiments, E-ISSN 1940-087X, no 140, article id e57877Article in journal (Refereed) Published
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.

Keywords
Biochemistry, Issue 140, Mitochondria, translation, ribosome, cryo-EM, protein synthesis, biochemistry
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-166868 (URN)10.3791/57877 (DOI)000456452800018 ()30346389 (PubMedID)
Available from: 2019-03-06 Created: 2019-03-06 Last updated: 2024-01-17Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-4656-3362

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