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ATPase-dependent role of the atypical kinase Rio2 on the evolving pre-40S ribosomal subunit

Nature Structural & Molecular Biology volume 19, pages 1316–1323 (2012)Cite this article

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Abstract

Ribosome synthesis involves dynamic association of ribosome-biogenesis factors with evolving preribosomal particles. Rio2 is an atypical protein kinase required for pre-40S subunit maturation. We report the crystal structure of eukaryotic Rio2–ATP–Mg2+ complex. The active site contains ADP-Mg2+ and a phosphoaspartate intermediate typically found in Na+, K+ and Ca2+ ATPases but not protein kinases. Consistent with this finding, ctRio2 exhibits a robust ATPase activity in vitro. In vivo, Rio2 docks on the ribosome, with its active site occluded and its flexible loop positioned to interact with the pre-40S subunit. Moreover, Rio2 catalytic activity is required for its dissociation from the ribosome, a necessary step in pre-40S maturation. We propose that phosphoryl transfer from ATP to Asp257 in Rio2's active site and subsequent hydrolysis of the aspartylphosphate could be a trigger to power late cytoplasmic 40S subunit biogenesis.

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Figure 1: Crystal structure of the ctRio2 kinase.
Figure 2: ctRio2 has ATPase activity in vitro.
Figure 3: Positioning of Rio2 kinase into the cryo-EM density map of the yeast pre-40S subunit.
Figure 4: Rio2's catalytic activity is required for 40S subunit biogenesis.
Figure 5: Rio2's kinase activity is required for its own recycling from the pre-40S subunit.

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  • 05 December 2012

    In the version of this supplementary file originally posted online, the labels for the chemicals shown in Supplementary Figure 5d contained errors. The errors have been corrected in this file 5 December 2012.

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Acknowledgements

We thank K. Shokat (Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco California, USA) for providing the ATP analog compounds 3-MB-PP1 and 1-NA-PP1; K. Karbstein (Scripps Research Institute, Jupiter, Florida, USA) for providing anti-Tsr1 antibody; S. Amlacher for providing C. thermophilum cDNA and E. Thomson and S. Griesel for providing ctHrr25 expression vector (Biochemistry Center, University of Heidelberg, Heidelberg, Germany); J. Lechner and his team for mass spectrometry, G. Lorimer (Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA) for assistance and advice in steady-state kinetic measurements, G. Manikas for assistance with single-turnover and ATP-binding assays and M. Gnädig for her excellent technical assistance. Data collection was conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, supported by grants from the US National Center for Research Resources (5P41RR015301-10) and the US National Institute of General Medical Sciences (8 P41 GM103403-10) from the US National Institutes of Health. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. This work was funded by the postdoctoral fellowship from the Medical Faculty of the University of Heidelberg to S.F.-C., the German Research Council (DFG Hu363/10-4) to E.H. and US National Institutes of Health National Cancer Institute grant (K22CA123152) to N.L.-L.

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Author notes

  1. Thorsten Schäfer

    Present address: Present address: Novartis Institutes for Biomedical Research, Basel, Switzerland.,

  2. Sébastien Ferreira-Cerca and Vatsala Sagar: These authors contributed equally to this work.

Authors and Affiliations

  1. Biochemistry Center, University of Heidelberg, Heidelberg, Germany

    Sébastien Ferreira-Cerca, Thorsten Schäfer, Anne-Maria Wesseling & Ed Hurt

  2. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA

    Vatsala Sagar, Momar Diop, Haiyun Lu, Eileen Chai & Nicole LaRonde-LeBlanc

  3. University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland, USA

    Nicole LaRonde-LeBlanc

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Contributions

S.F.-C., T.S., N.L.-L. and E.H. conceived of the experiments. S.F.-C., T.S. and A.-M.W. constructed all plasmids and yeast strains and carried out all yeast genetic experiments. S.F.-C. performed all the sucrose-gradient analyses, tandem-affinity purifications of yeast proteins and single-turnover and nucleotide-binding analyses. T.S. performed in vitro phosphorylation experiments on purified pre-40S. S.F.-C. and A.-M.W. performed the biochemical characterization of ctRio2. V.S. and E.C. optimized and performed protein purification and identified and refined crystallization conditions. V.S. and N.L.-L. determined the crystal structures. M.D. performed hydroxylamine phosphate release assays and steady-state rate determination using purified protein provided by H.L. N.L.-L. performed ctRio2-40S docking analysis. E.H. and N.L.-L. supervised the work; S.F.-C., N.L.-L. and E.H. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Ed Hurt or Nicole LaRonde-LeBlanc.

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Supplementary Figures 1–5 and Supplementary Tables 1–2 and Supplementary Note (PDF 6835 kb)

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Ferreira-Cerca, S., Sagar, V., Schäfer, T. et al. ATPase-dependent role of the atypical kinase Rio2 on the evolving pre-40S ribosomal subunit. Nat Struct Mol Biol 19, 1316–1323 (2012). https://doi.org/10.1038/nsmb.2403

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