Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Structural and mechanistic insights into an archaeal DNA-guided Argonaute protein

Nature Microbiology volume 2, Article number: 17035 (2017) Cite this article

Subjects

Abstract

Argonaute (Ago) proteins in eukaryotes are known as key players in post-transcriptional gene silencing1, while recent studies on prokaryotic Agos hint at their role in the protection against invading DNA2,3. Here, we present crystal structures of the apo enzyme and a binary Ago-guide complex of the archaeal Methanocaldococcus jannaschii (Mj) Ago. Binding of a guide DNA leads to large structural rearrangements. This includes the structural transformation of a hinge region containing a switch helix, which has been shown for human Ago2 to be critical for the dynamic target search process4–6. To identify key residues crucial for MjAgo function, we analysed the effect of several MjAgo mutants. We observe that the nature of the 3′ and 5′ nucleotides in particular, as well as the switch helix, appear to impact MjAgo cleavage activity. In summary, we provide insights into the molecular mechanisms that drive DNA-guided DNA silencing by an archaeal Ago.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overall folding topology of MjAgo and guide 5′-nucleotide preference.
Figure 2: Crystal structure of the binary MjAgo guide–DNA complex.
Figure 3: Guide-DNA recognition by the MjAgo PAZ domain.
Figure 4: Structural differences between apo MjAgo and the MjAgo binary complex.

Similar content being viewed by others

References

  1. Liu, J . et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    Article  CAS  Google Scholar 

  2. Olovnikov, I., Chan, K., Sachidanandam, R., Newman, D. K. & Aravin, A. A. Bacterial Argonaute samples the transcriptome to identify foreign DNA. Mol. Cell 51, 594–605 (2013).

    Article  CAS  Google Scholar 

  3. Sheng, G. et al. Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proc. Natl Acad. Sci. USA 111, 652–657 (2014).

    Article  CAS  Google Scholar 

  4. Chandradoss, S. D., Schirle, N. T., Szczepaniak, M., MacRae, I. J. & Joo, C. A dynamic search process underlies microRNA targeting. Cell 162, 96–107 (2015).

    Article  CAS  Google Scholar 

  5. Elkayam, E . et al. The structure of human Argonaute-2 in complex with miR-20a. Cell 150, 100–110 (2012).

    Article  CAS  Google Scholar 

  6. Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012).

    Article  CAS  Google Scholar 

  7. Swarts, D. C. et al. The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol. 21, 743–753 (2014).

    Article  CAS  Google Scholar 

  8. Nakanishi, K., Weinberg, D. E., Bartel, D. P. & Patel, D. J. Structure of yeast Argonaute with guide RNA. Nature 486, 368–374 (2012).

    Article  CAS  Google Scholar 

  9. Deerberg, A., Willkomm, S. & Restle, T. Minimal mechanistic model of siRNA-dependent target RNA slicing by recombinant human Argonaute 2 protein. Proc. Natl Acad. Sci. USA 110, 17850–17855 (2013).

    Article  CAS  Google Scholar 

  10. Nakanishi, K. et al. Eukaryote-specific insertion elements control human Argonaute slicer activity. Cell Rep. 3, 1893–1900 (2013).

    Article  CAS  Google Scholar 

  11. Schirle, N. T., Sheu-Gruttadauria, J., Chandradoss, S. D., Joo, C. & MacRae, I. J. Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets. eLife 4, e07646 (2015).

  12. Wang, Y . et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754–761 (2009).

    Article  CAS  Google Scholar 

  13. Lambert, N. J., Gu, S. G. & Zahler, A. M. The conformation of microRNA seed regions in native microRNPs is prearranged for presentation to mRNA targets. Nucleic Acids Res. 39, 4827–4835 (2011).

    Article  CAS  Google Scholar 

  14. Wee, L. M., Flores-Jasso, C. F., Salomon, W. E. & Zamore, P. D. Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151, 1055–1067 (2012).

    Article  CAS  Google Scholar 

  15. Parker, J. S., Parizotto, E. A., Wang, M., Roe, S. M. & Barford, D. Enhancement of the seed-target recognition step in RNA silencing by a PIWI/MID domain protein. Mol. Cell 33, 204–214 (2009).

    Article  CAS  Google Scholar 

  16. Swarts, D. C. et al. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA. Nucleic Acids Res. 43, 5120–5129 (2015).

    Article  CAS  Google Scholar 

  17. Jones, W. J., Leigh, J. A., Mayer, F., Woese, C. R. & Wolfe, R. S. Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent. Arch. Microbiol. 136, 254–261 (1983).

    Article  CAS  Google Scholar 

  18. Kaya, E. et al. A bacterial Argonaute with noncanonical guide RNA specificity. Proc. Natl Acad. Sci. USA 113, 4057–4062 (2016).

    Article  CAS  Google Scholar 

  19. Zander, A., Holzmeister, P., Klose, D., Tinnefeld, P. & Grohmann, D. Single-molecule FRET supports the two-state model of Argonaute action. RNA Biol. 11, 45–56 (2014).

    Article  CAS  Google Scholar 

  20. Parker, J. S., Roe, S. M. & Barford, D. Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex. Nature 434, 663–666 (2005).

    Article  CAS  Google Scholar 

  21. Yuan, Y. R. et al. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005).

    Article  CAS  Google Scholar 

  22. Willkomm, S., Zander, A., Grohmann, D. & Restle, T. Mechanistic insights into archaeal and human Argonaute substrate binding and cleavage properties. PLoS ONE 11, e0164695 (2016).

    Article  Google Scholar 

  23. Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).

    Article  CAS  Google Scholar 

  24. Schirle, N. T., Sheu-Gruttadauria, J. & MacRae, I. J. Structural basis for microRNA targeting. Science 346, 608–613 (2014).

    Article  CAS  Google Scholar 

  25. Zhu, L . et al. A flexible domain–domain hinge promotes an induced-fit dominant mechanism for the loading of guide-DNA into Argonaute protein in Thermus thermophilus. J. Phys. Chem. B 120, 2709–2720 (2016).

    Article  CAS  Google Scholar 

  26. Frank, F., Hauver, J., Sonenberg, N. & Nagar, B. Arabidopsis Argonaute MID domains use their nucleotide specificity loop to sort small RNAs. EMBO J. 31, 3588–3595 (2012).

    Article  CAS  Google Scholar 

  27. Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862 (2001).

    Article  CAS  Google Scholar 

  28. Frank, F., Sonenberg, N. & Nagar, B. Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465, 818–822 (2010).

    Article  CAS  Google Scholar 

  29. Millhauser, G. L. Views of helical peptides: a proposal for the position of 310-helix along the thermodynamic folding pathway. Biochemistry 34, 3873–3877 (1995).

    Article  CAS  Google Scholar 

  30. Kwak, P. B. & Tomari, Y. The N domain of Argonaute drives duplex unwinding during RISC assembly. Nat. Struct. Mol. Biol. 19, 145–151 (2012).

    Article  CAS  Google Scholar 

  31. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010).

    Article  CAS  Google Scholar 

  32. Gildea, R. J. et al. New methods for indexing multi-lattice diffraction data. Acta Crystallogr. D 70, 2652–2666 (2014).

    Article  CAS  Google Scholar 

  33. Diederichs, K. & Karplus, P. A. Better models by discarding data? Acta Crystallogr. D 69, 1215–1222 (2013).

    Article  CAS  Google Scholar 

  34. Evans, P. Biochemistry. Resolving some old problems in protein crystallography . Science 336, 986–987 (2012).

    Article  CAS  Google Scholar 

  35. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    Article  CAS  Google Scholar 

  36. Skubak, P. & Pannu, N. S. Automatic protein structure solution from weak X-ray data. Nat. Commun. 4, 2777 (2013).

    Article  Google Scholar 

  37. Cowtan, K. Fitting molecular fragments into electron density. Acta Crystallogr. D 64, 83–89 (2008).

    Article  CAS  Google Scholar 

  38. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  39. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  40. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  41. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    Article  CAS  Google Scholar 

  42. Blanc, E . et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004).

    Article  CAS  Google Scholar 

  43. Painter, J. & Merritt, E. A. TLSMD web server for the generation of multi-group TLS models. J. Appl. Crystallogr. 39, 109–111 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  45. Chen, V. B. et al. Molprobity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  46. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268 (2004).

    Article  CAS  Google Scholar 

  47. Larkin, M. A. et al. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  Google Scholar 

  48. Pei, J., Tang, M. & Grishin, N. V. PROMALS3D web server for accurate multiple protein sequence and structure alignments. Nucleic Acids Res. 36, W30–W34 (2008).

    Article  CAS  Google Scholar 

  49. Taylor, D., Cawley, G. & Hayward, S. Quantitative method for the assignment of hinge and shear mechanism in protein domain movements. Bioinformatics 30, 3189–3196 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG grant no. GR 3840/2-1 to D.G.), the Excellence Cluster CIPSM and Fonds der Chemischen Industrie (to S.S.). The authors thank the ESRF, SLS and DESY for beamtime and the staff of beamlines ID23-1, ID23-2, ID29 (ESRF), PX I (SLS) and PETTRA IV (DESY) for assistance with crystal testing and data collection. The authors thank A. Lebedev for discussions and R. Sterner for providing access to a FP-600 spectrometer, which allowed us to perform anisotropy measurements.

Author information

Author notes

  1. Sarah Willkomm and Christine A. Oellig: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Microbiology, University of Regensburg, 93053 Regensburg, Germany

    Sarah Willkomm, Adrian Zander & Dina Grohmann

  2. Department of Chemistry, Center for Integrated Protein Science Munich CIPSM, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany

    Christine A. Oellig & Sabine Schneider

  3. Institute for Molecular Medicine, University of Lübeck, 23538 Lübeck, Germany

    Tobias Restle

  4. CCP4, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, OX11 0FA, Didcot, UK

    Ronan Keegan

  5. Institute of Integrative Biology, University of Liverpool, L69 7ZB, Liverpool, UK

    Ronan Keegan

Authors
  1. Sarah Willkomm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christine A. Oellig
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adrian Zander
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tobias Restle
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ronan Keegan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dina Grohmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sabine Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.W. generated, expressed and purified MjAgo mutants, and conducted activity assays and fluorescence titration experiments. C.A.O. expressed, purified and crystallized MjAgo. A.Z. expressed and purified MjAgo and conducted activity assays. R.K. provided critical input for structure determination. S.S. derivatized crystals, collected crystallographic data, and determined and built the structures. S.W., T.R., D.G. and S.S. analysed the data. S.W. and S.S. prepared figures. S.W., D.G. and S.S. designed the research project and wrote the manuscript. All authors edited the manuscript.

Corresponding authors

Correspondence to Dina Grohmann or Sabine Schneider.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-12, Supplementary Tables 1 and 2, and Supplementary References. (PDF 2764 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Willkomm, S., Oellig, C., Zander, A. et al. Structural and mechanistic insights into an archaeal DNA-guided Argonaute protein. Nat Microbiol 2, 17035 (2017). https://doi.org/10.1038/nmicrobiol.2017.35

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.35

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing