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

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 process46. 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.

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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.

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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.

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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.

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Correspondence to Dina Grohmann or Sabine Schneider.

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The authors declare no competing financial interests.

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

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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

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