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.
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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.
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Integrated supplementary information
Supplementary Figure 1 8-Azanebularine-containing duplex RNAs form a tight and specific complex with hADAR2d.
(a) Representative electrophoretic mobility shift assay (EMSA) gel displaying tight and specific binding of hADARd E488Q mutant and N-containing hGLI1 24mer duplex. Lane 1: no protein added; Lanes 2-11: 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 nM hADAR2d E488Q. (b) Fitted plot of fraction RNA bound vs. hADAR2d E488Q concentration.
Supplementary Figure 2 Overall structure of the hADAR2d–RNA complex.
(a) (Left) A simulated-annealed composite omit electron density map calculated in PHENIX (Afonine, P.V. et al. Acta Crystallogr D Biol Crystallogr 71, 646-666, 2015) is shown in blue mesh. The map is contoured at 1σ at 2.75Å resolution. The inositol hexakisphosphate is shown in white-colored-carbon sticks, and the active site zinc atom as a gray sphere near the flipped out base. (Right) View of hADAR2d E488Q-Bdf2 complex showing protein surface electrostatic potential. The electrostatic potential was calculated use the Adaptive Poisson-Boltzmann Solver (APBS) plugin in PyMol (Baker, N.A et al. Proc Natl Acad Sci USA 98, 10037-10041, 2001). Electrostatic potential was calculated using the default values, and displayed on the protein surface with blue and red representing the positive and negative potentials respectively. (b) Summary of protein-RNA contacts observed in the Gli1-hADAR2d E488Q complex. (c) Summary of protein-RNA contacts observed in the hADAR2d WT-Bdf2-C complex. (d) Summary of protein-RNA contacts observed in the hADAR2d WT-Bdf2-U complex.
Supplementary Figure 3 Close-up views of the active site and 5′-nearest-neighbor nucleotides with electron density map.
(a) (Left) Close-up view of the flipped out base with electron density. The flipped out 8-azanebularine nucleotide (hADAR2d E488Q + Bdf2-C) is shown as sticks with white-colored carbon atoms. Residues that ligate the active site zinc are shown with brown-colored carbon atoms. Simulated annealed composite omit map shown in blue mesh contoured at 1σ. Numbers next to yellow dashed lines represent ligation distances in Å. (Right) Simulated annealed omit map of the flipped out base. The 8-azanebularine nucleotide was omitted from the structure and a simulated annealed omit map calculated in PHENIX (Adams, P.D et al. Acta Crystallogr D Biol Crystallogr 66, 213-221, 2010). Positive electron density contoured at 3σ is displayed in green. This clearly shows the presence of oxygen O6 (extending from carbon 6) ligating to the active site zinc atom. This definitively reveals the hydrated intermediate is being trapped in the crystal. (b) Close up view of 5’ UA pair from (hADARd WT + Bdf2-U) shown with composite omit-map electron density at 1σ. The geometry of the 5’ nearest neighbor base pair from hADAR2d WT+ Bdf2-U (sticks) and electron density overlaid with structure of hADAR2d WT+ Bdf2-C (lines) suggests a non-canonical base pair between the A and U.
Supplementary Figure 4 Intercalating residue and adjacent RNA.
(a) Overlay of intercalating residue and RNA from three structures. Overlay of structures of wild type and E488Q hADAR2d with Bdf2-C RNA and wild type hADAR2d with Bdf2-U RNA show high degree of similarity in both RNA and enzyme conformations (b) Comparison of ADAR2 and HhaI Mtase orphan base recognition. Shown is the interaction between intercalating residue side chain and orphan nucleotide in ADAR2 (Left) and HhaI cytidine methyltransferase (Right). For ADAR2 the side chain approaches the nucleotide from the minor groove of the A form helix while for HhaI Mtase the approach is made from the major groove of the B form DNA (Klimasauskas, S. et al. Cell, 76, 357-369 1994).
Supplementary Figure 5 Comparison of protein–nucleic acid complexes with flipped nucleotides.
(a) Structures of DNA modifying enzymes with base flipping dependent mechanisms approaching from the minor groove. Protein in blue, DNA in yellow, which is bent, flipped nucleotide in red. (b) Structures of protein–RNA complexes with flipped nucleotides. Protein in blue, RN in pink, flipped nucleotide in red.
Supplementary Figure 6 Access of residue 488 into the minor grooves of A-form and B-form helices.
Overlay of intercalating residue 488 with idealized A form RNA (Left) shows that the geometry of the minor groove in an A form helix allows the side chain to occupy the position of the adenosine base (yellow). Overlay of residue 488 with idealized B form DNA (Right) shows that the greater depth of the minor groove in a B form helix prevents the side chain from intercalating into the space occupied by an adenosine base (yellow). Idealized A-RNA and B-DNA of Bdf2 sequence were superimposed onto the RNA duplex observed in the hADAR2d complexed with RNA (as shown in Figure 4B).
Supplementary Figure 7 Nearest-neighbor effects of hADAR2d.
(a) Predicted secondary structure of GLI1 mRNA editing site showing 3’ nearest neighbor and complementary mutations. Edited A is highlighted in red. (b) Comparison of deamination rates in 3’ nearest neighbor mutants in hGLI1 mRNA. Observed in vitro deamination rate constants for deamination of hGLI1 mRNA by hADAR2d and 3’ nearest neighbor mutants.
(c) Space filling models of 5’ nearest neighbor base pair and hADAR2d G489. Minor groove edge of U11-A13’ base pair is in close proximity to the protein backbone at G489 in the Bdf2-hADAR2d complex (top left). U11 is located on the 5’ side of the editing site (i.e. 5’ U). This site appears to accommodate a 5’ A (top right) but when either a C-G (bottom left) or a G-C (bottom right) base pair is modeled into this site, a clash is apparent between the G 2-amino group and the alpha carbon of G489.
Supplementary Figure 8 hADAR1 residues associated with AGS, mapped to analogous positions in hADAR2d.
(a) Space filling model of hADAR2d E488Q + Bdf2-C RNA highlighting the locations corresponding to residues in hADAR1 where mutations cause Aicardi–Goutières syndrome. Modeling positions of surface residues of hADAR1 with mutations previously associated with Aicardi–Goutières syndrome (Rice, G.I. et al. Nat Genetics, 44, 1243-1248, 2012) provides insight into the basis for the mutations’ effect on editing. hADAR2 residues corresponding to hADAR1 R892 and G1007 (K376 and G487 respectively) are found at the protein/RNA binding interface. K999 maps to ADAR2’s 5’ major groove binding loop (Q479). While Y1112 and D1113 are not directly involved in the protein/RNA interface, they appear to be in position to stabilize an RNA binding loop. E588 in hADAR2 (corresponding to D1113 in hADAR1) is involved in an intramolecular salt bridge with highly conserved R349 located adjacent to RNA contact residue R348. (b) hADAR2 G487 is located at the protein-RNA interface. Mutation to arginine (corresponding to the G1007R mutation in hADAR1 associated with the human diseases AGS and DHS) would cause a severe clash with the RNA, especially with orphan C11’ and A12’. The G487R mutation is shown with transparent CPK spheres with magenta-colored carbons.
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Supplementary Text and Figures
Supplementary Figures 1–8 and Supplementary Tables 1 and 2 (PDF 3473 kb)
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). (MOV 1557 kb)
hADAR2 morph
This file contains a morphing movie of the change in hADAR2d conformation before and after RNA binding. (MOV 988 kb)
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Matthews, M., Thomas, J., Zheng, Y. et al. Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat Struct Mol Biol 23, 426–433 (2016). https://doi.org/10.1038/nsmb.3203
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DOI: https://doi.org/10.1038/nsmb.3203
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