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Cryo-EM and Computational Biology of Macromolecular Complexes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Sanyal)ORCID iD: 0000-0002-0908-9924
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

The ribosome is a large, ancient multicomponent macromolecular complex which is highly amenable to study by cryogenic electron microscopy (cryo-EM) and computation biology methods. This thesis delves into the structure of both prokaryotic and eukaryotic ribosomes in the context of determining a solution to emerging antimicrobial resistance. We show that thermorubin (THB) binds to the E. coli ribosome at intersubunit bridge B2a, flipping out 23S rRNA residue C1914 which interferes with A-site substrates. The position and rearrangements caused by THB also accounts for the biochemical results showing a decrease in elongation, termination and recycling phases of translation. Also using cryo-EM we looked at the Giardia intestinalis ribosome, determining six high-resolution structures representing translocation intermediates. Giardia is a protozoan parasite causing diarrhoea in humans, with metronidazole strains emerging. As the ribosome is often a target for antimicrobial drugs, work on the structure and function of the ribosome is of utmost important in determining an alternative therapeutic approach to the treatment of giardiasis. We also show naturally bound tRNAs and eEF2 on the Giardia ribosome, exhibiting eukaryote-specific subunit rolling and eEF2 with GDP in a uniquely positioned Pi primed for release, adding to the mechanism of translocation in protists as well as illustrating the evolution of both the structure and function of translation machinery. Finally, the molecular basis of thermostability in translational GTPases is explored using molecular dynamics of mesophilic and thermophilic elongation factor EF-Tu. Through ancestral sequence reconstruction two key interactions: in the GTPase domain; and an interdomain interaction were shown to be important in the overall structural stability of EF-Tu in high temperature environments. These studies together highlight the strength of utilising both structural and computational techniques to explore the translation apparatus.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2023. , p. 45
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 2232
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-495335ISBN: 978-91-513-1698-7 (print)OAI: oai:DiVA.org:uu-495335DiVA, id: diva2:1731371
Public defence
2023-03-17, A1:107a, Biomedicinskt centrum, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2023-02-24 Created: 2023-01-26 Last updated: 2023-02-24
List of papers
1. Insights into Translocation Mechanism and Ribosome Evolution from Cryo-EM Structures of Translocation Intermediates of Giardia intestinalis
Open this publication in new window or tab >>Insights into Translocation Mechanism and Ribosome Evolution from Cryo-EM Structures of Translocation Intermediates of Giardia intestinalis
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Giardia intestinalis is a protozoan parasite that causes diarrhea in humans. Using single-particle cryo-Electron Microscopy, we have determined high-resolution structures of six naturally populated translocation intermediates, from ribosomes isolated directly from actively growing Giardia cells. The highly compact and uniquely GC-rich Giardia ribosomes possess eukaryotic rRNAs and ribosomal-proteins, but retain some bacterial features. The translocation intermediates, with naturally-bound tRNAs and eEF2, display characteristic ribosomal intersubunit rotation and small subunit’s head swiveling - universal for translocation. In addition, we observe the eukaryote-specific ‘subunit rolling’ dynamics, albeit with limited features. Finally, the eEF2•GDP state features a uniquely positioned ‘leaving Pi’ that proposes hitherto unknown molecular events of Pi- and eEF2 release from the ribosome at the final stage of translocation. In summary, our study elucidates the mechanism of translocation in the protists and illustrates evolution of the translation machinery from bacteria to eukaryotes both from the structural and mechanistic perspectives.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-495331 (URN)
Available from: 2023-01-26 Created: 2023-01-26 Last updated: 2023-01-26
2. Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria
Open this publication in new window or tab >>Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 918Article in journal (Refereed) Published
Abstract [en]

Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.

Place, publisher, year, edition, pages
Springer Nature, 2023
Keywords
Antibiotic, Thermorubin, Ribosome, Translation inhibition, Subunit tethering
National Category
Biochemistry Molecular Biology Structural Biology
Research subject
Biochemistry; Biology with specialization in Molecular Biology; Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-486747 (URN)10.1038/s41467-023-36528-7 (DOI)001001567400001 ()36806263 (PubMedID)
Funder
Uppsala University
Available from: 2022-10-19 Created: 2022-10-19 Last updated: 2025-02-20Bibliographically approved
3. Crystal Structures of Ancestral Orthologues Reveal the Molecular Basis of Thermostability in Thermophilic EF-Tus
Open this publication in new window or tab >>Crystal Structures of Ancestral Orthologues Reveal the Molecular Basis of Thermostability in Thermophilic EF-Tus
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The molecular basis of protein thermostability is diverse and unclear. To better understand it, we solved high-resolution crystal structures of four 0.5 – 3.5 billion year old ancestral bacterial Elongation Factor-Tus (EF-Tu). Structural comparison revealed two key interactions, unique for the thermophilic EF-Tus; i) a hydrogen bond between a G-domain tyrosine and the α- phosphate of the guanine nucleotide that stabilizes GTP/GDP; and ii) an inter-domain salt-bridge, tethering the Domains II and III via an arginine and a glutamic acid, respectively. We could reverse the thermostability profiles of the thermophilic and mesophilic EF-Tus by adding or removing these interactions, which were validated with aggregation, biophysical, and functional assays. Further, molecular dynamics simulations demonstrated that these interactions contribute to thermostability via the Mg2+ in the nucleotide binding site. Furthermore, the inter-domain interaction restricts the transition between the open and the closed conformations of the EF-Tus, thereby regulating their thermoactivity. 

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-495334 (URN)
Available from: 2023-01-26 Created: 2023-01-26 Last updated: 2023-01-26

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