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Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity

Nature Structural & Molecular Biology volume 23, pages 426433 (2016) | Download Citation

Abstract

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

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Acknowledgements

The authors acknowledge funding from the US National Institutes of Health (NIH) grant R01GM061115 (P.A.B.). A.I.S. was supported by NIH training grant T32 GM007377. C. Palumbo is acknowledged for technical assistance. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research, and by the NIH, US National Institute of General Medical Sciences (including P41GM103393). Part of this work is also based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (P41 GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Author information

Author notes

    • Melissa M Matthews
    •  & Justin M Thomas

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry, University of California, Davis, Davis, California, USA.

    • Melissa M Matthews
    • , Justin M Thomas
    • , Yuxuan Zheng
    • , Kiet Tran
    • , Kelly J Phelps
    • , Jocelyn Havel
    • , Andrew J Fisher
    •  & Peter A Beal
  2. Department of Molecular and Cellular Biology, University of California, Davis, Davis, California, USA.

    • Anna I Scott
    •  & Andrew J Fisher

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Contributions

J.M.T., M.M.M., A.I.S., and Y.Z. purified protein. K.J.P. and J.M.T. designed and purified RNA for crystallography and characterized protein-RNA binding. M.M.M. and A.I.S. conducted crystallization trials. M.M.M. and A.J.F. collected diffraction data and solved and refined the crystal structures. J.M.T., Y.Z., and J.H. measured enzyme reaction rates. K.T. synthesized 8-azanebularane phosphoramidite. J.M.T. and A.I.S. conducted mutagenesis. J.M.T., M.M.M., P.A.B. and A.J.F. analyzed the structures. P.A.B. wrote the initial manuscript draft. J.M.T., M.M.M., P.A.B., and A.J.F. edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Andrew J Fisher or Peter A Beal.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1 and 2

Videos

  1. 1.

    RNA morph

    This file contains a morphing movie of the change in conformation of the RNA before and after hADAR2d binding (assuming an ideal A-form duplex RNA starting point).

  2. 2.

    hADAR2 morph

    This file contains a morphing movie of the change in hADAR2d conformation before and after RNA binding.

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DOI

https://doi.org/10.1038/nsmb.3203

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