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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 13161323 (2012) | Download Citation

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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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  • 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.


Primary accessions

Referenced accessions

Electron Microscopy Data Bank


  1. 1.

    , , , & Rio2p, an evolutionarily conserved, low abundant protein kinase essential for processing of 20 S pre-rRNA in Saccharomyces cerevisiae. J. Biol. Chem. 278, 22537–22545 (2003).

  2. 2.

    , , & Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae. Mol. Cell Biol. 23, 2083–2095 (2003).

  3. 3.

    , , , & The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J. 22, 1370–1380 (2003).

  4. 4.

    et al. The post-transcriptional steps of eukaryotic ribosome biogenesis. Cell Mol. Life Sci. 65, 2334–2359 (2008).

  5. 5.

    , , & Ribosome assembly in eukaryotes. Gene 313, 17–42 (2003).

  6. 6.

    et al. Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2. J. Cell Biol. 185, 1167–1180 (2009).

  7. 7.

    , , & Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking. EMBO J. 29, 2026–2036 (2010).

  8. 8.

    et al. Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates. Science 333, 1449–1453 (2011).

  9. 9.

    , , & Autophosphorylation of Archaeoglobus fulgidus Rio2 and crystal structures of its nucleotide-metal ion complexes. FEBS J. 272, 2800–2810 (2005).

  10. 10.

    & Crystal structure of A. fulgidus Rio2 defines a new family of serine protein kinases. Structure 12, 1585–1594 (2004).

  11. 11.

    et al. Insight into structure and assembly of the nuclear pore complex by utilizing the genome of a eukaryotic thermophile. Cell 146, 277–289 (2011).

  12. 12.

    & Evidence for an aspartyl phosphate residue at the active site of sodium and potassium ion transport adenosine triphosphatase. J. Biol. Chem. 248, 6993–7000 (1973).

  13. 13.

    Biology, structure and mechanism of P-type ATPases. Nat. Rev. Mol. Cell Biol. 5, 282–295 (2004).

  14. 14.

    , & The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52 (1988).

  15. 15.

    & Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem. Sci. 36, 65–77 (2011).

  16. 16.

    , , , & Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY. J. Biol. Chem. 264, 21770–21778 (1989).

  17. 17.

    , , , & A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif. J. Biol. Chem. 273, 14107–14112 (1998).

  18. 18.

    et al. Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry 32, 2154–2161 (1993).

  19. 19.

    & Designing bisubstrate analog inhibitors for protein kinases. Pharmacol. Ther. 93, 145–157 (2002).

  20. 20.

    et al. Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature 441, 651–655 (2006).

  21. 21.

    & On the attachment of the nuclear pore complex. J. Cell Biol. 62, 746–754 (1974).

  22. 22.

    et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

  23. 23.

    , , , & Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16, 673–683 (2008).

  24. 24.

    , & Magic bullets for protein kinases. Trends Cell Biol. 11, 167–172 (2001).

  25. 25.

    & P-type ATPases. Annu. Rev. Biophys. 40, 243–266 (2011).

  26. 26.

    , , , & Crystal structure of the alpha-kinase domain of Dictyostelium myosin heavy chain kinase A. Sci. Signal. 3, ra17 (2010).

  27. 27.

    , , , & Crystal structures of c-Src reveal features of its autoinhibitory mechanism. Mol. Cell 3, 629–638 (1999).

  28. 28.

    et al. Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell 112, 859–871 (2003).

  29. 29.

    et al. Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2. Nat. Struct. Mol. Biol. 15, 112–113 (2008).

  30. 30.

    et al. The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334, 1524–1529 (2011).

  31. 31.

    et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004).

  32. 32.

    et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

  33. 33.

    et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

  34. 34.

    , , , & Role of SRP RNA in the GTPase cycles of Ffh and FtsY. Biochemistry 40, 15224–15233 (2001).

  35. 35.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  36. 36.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  37. 37.

    The Pymol molecular graphics system (Delano Scientific, 2002).

  38. 38.

    , & VMD: visual molecular dynamics. J. Mol. Graph 14, 33–38, 27–28 (1996).

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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.

Author information

Author notes

    • Thorsten Schäfer

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

    • Sébastien Ferreira-Cerca
    •  & Vatsala Sagar

    These authors contributed equally to this work.


  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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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ed Hurt or Nicole LaRonde-LeBlanc.

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