In Vitro Reconstitution of the S. aureus 50S Ribosomal Subunit and GTP-Binding Factor YSXC Complex for Structural Studies

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Abstract

The proper assembly and maturation of ribosomal subunits are critical processes that ensure the functional activity, translation efficiency, and fidelity of the ribosome. One of the protein factors involved in the processes of ribosome large subunit maturation is the GTP-binding protein YsxC, which is found in many bacteria. In the absence of YsxC, immature ribosomal intermediates, designated 45S subunits, are observed to accumulate within the cell. These are unable to associate with the small subunit of the ribosome and thus fail to form ribosomes capable of carrying out the necessary functions. The deletion of the ysxC gene is lethal to S. aureus. The mechanism of YsxC interaction with the Staphylococcus aureus ribosome remains to be elucidated. In this study, we devised a protocol for the isolation, purification, and assembly of the YsxC protein complex with the 50S subunit of the Staphylococcus aureus ribosome, which enabled us to obtain a sample suitable for registration of transmission cryo-electron microscopy data.

About the authors

A. D. Biktimirov

Kazan Federal University

Kazan, 420008 Russia

M. M. Yusupov

Kazan Federal University; National Research Center “Kurchatov Institute”; Institute of Genetics, Molecular and Cellular Biology, CNRS UMR7104, INSERM U964

Kazan, 420008 Russia; Moscow, 123182 Russia; Universit’e de Strasbourg, Illkirch, F‑67400 France

K. S. Usachev

Kazan Federal University

Email: konstantin.usachev@kpfu.ru
Kazan, 420008 Russia

References

  1. GBD2021 Antimicrobial Resistance Collaborators. (2024) Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. Lancet. 404, 1199–1226.
  2. Foster T.J. (2017) Antibiotic resistance in Staphylococcus aureus. Current status and future prospects. FEMS Microbiol. Rev. 41, 430–449.
  3. Vestergaard M., Frees D., Ingmer H. (2019) Antibiotic resistance and the MRSA problem. Microbiol. Spectr. 7, 1–23
  4. Юсупова Г.Ж., Юсупов М.М. (2021) Путь к расшифровке атомных структур прокариатической и эукариотической рибосом (обзор). Биохимия. 86, 1120–1137.
  5. Seffouh A., Nikolay R., Ortega J. (2024) Critical steps in the assembly process of the bacterial 50S ribosomal subunit. Nucl. Acids Res. 52, 4111–4123.
  6. Maksimova E., Kravchenko O., Korepanov A., Stolboushkina E. (2022) Protein assistants of small ribosomal subunit biogenesis in bacteria. Microorganisms. 10, 747.
  7. Shajani Z., Sykes M.T., Williamson J.R. (2011) Assembly of bacterial ribosomes. Annu. Rev. Biochem. 80, 501–526.
  8. Schaefer L., Uicker W.C., Wicker-Planquart C., Foucher A.E., Jault J.M., Britton R.A. (2006) Multiple GTPases participate in the assembly of the large ribosomal subunit in Bacillus subtilis. J. Bacteriol. 188, 8252–8258.
  9. Ni X., Davis J.H., Jain N., Razi A., Benlekbir S., McArthur A.G., Rubinstein J.L., Britton R.A., Williamson J.R., Ortega J. (2016) YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit. Nucl. Acids Res. 44, 8442–8455.
  10. Wicker-Planquart C., Foucher A.-E., Louwagie M., Britton R.A., Jault J.-M. (2008) Interactions of an essential Bacillus subtilis GTPase, YsxC, with ribosomes. J. Bacteriol. 190, 681–690.
  11. Goyal A., Muthu K., Panneerselvam M., Pole A.K., Ramadas K. (2011) Molecular dynamics simulation of the Staphylococcus aureus YsxC protein: molecular insights into ribosome assembly and allosteric inhibition of the protein. J. Mol. Model. 17, 3129–3149.
  12. Биктимиров А.Д., Исламов Д.Р., Валидов Ш.З., Петерс Г.С., Халиуллина А.В., Юсупов М.М., Усачев К.С. (2023) Выделение, очистка и анализ методом малоуголового рентгеновского рассеяния GTPазы YsxC из золотистого стафилококка. Кристаллография. 68, 204–208.
  13. Biktimirov A., Islamov D., Fatkhullin B., Lazarenko V., Validov S., Yusupov M., Usachev K. (2024) Crystal structure of GTPase YsxC from Staphylococcus aureus. Biochem. Biophys. Res. Commun. 699, 149545.
  14. Golubev A., Fatkhullin B., Khusainov I., Jenner L., Gabdulkhakov A., Validov S., Yusupova G., Yusupov M., Usachev K. (2020) Cryo-EM structure of the ribosome functional complex of the human pathogen Staphylococcus aureus at 3.2 Å resolution. FEBS Lett. 594, 3551–3567.
  15. Garaeva N., Fatkhullin B., Murzakhanov F., Gafurov M., Golubev A., Bikmullin A., Glazyrin M., Kieffer B., Jenner L., Klochkov V., Aganov A., Rogachev A., Ivankov O., Validov Sh., Yusupov M., Usachev K. (2024) Structural aspects of RimP binding on small ribosomal subunit from Staphylococcus aureus. Structure. 32, 74–82.
  16. Бикмуллин А.Г., Нуруллина Л.И., Гараева Н.С., Клочква Э.А., Блохин Д.С., Голубев А.А., Валидов Ш.З., Хусаинов И.Ш., Усачев К.С., Юсупов М.М. (2020) Сборка комплекса 30S субъединицы рибосомы и фактора RbfA S. aureus in vitro для структурных исследований. Биохимия. 85, 637–646.
  17. Wicker-Planquart C., Ceres N., Jault J.-M. (2015) The C-terminal α-helix of YsxC is essential for its binding to 50S ribosome and rRNAs. FEBS Lett. 589, 2080–2086.
  18. Rozov A., Khusainov I., El Omari K., Duman R., Mykhaylyk V., Yusupov M., Westhof E., Wagner A., Yusupova G. (2019) Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction. Nat. Commun. 10, 2519.
  19. Khusainov I., Fatkhullin B., Pellegrino S., Bikmullin A., Validov Sh., Liu W., Gabdulkhakov A., Al Shebel A., Golubev A., Zeyer D., Trachtmann N., Sprenger G.A., Validov Sh., Usachev K., Yusupova G., Yusupov M. (2020) Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach. Nat. Commun. 11, 1656.

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