1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets

  1. Nicole T Schirle
  2. Jessica Sheu-Gruttadauria
  3. Stanley D Chandradoss
  4. Chirlmin Joo  Is a corresponding author
  5. Ian J MacRae  Is a corresponding author
  1. The Scripps Research Institute, United States
  2. Delft University of Technology, Netherlands
Research Article
Cite this article as: eLife 2015;4:e07646 doi: 10.7554/eLife.07646
9 figures and 3 tables

Figures

Structure of the t1 nucleotide binding pocket.

(A) Linear schematic of the Argonaute2 (Ago2) primary structure. (B) Crystal structure of Ago2 bound to a guide RNA (red) and target RNA bearing t1A nucleotide (blue; PDB ID: 4W5O). (C) Close-up view of the t1-binding pocket. Ordered water molecules shown as pink spheres. Protein shown in stick and surface representations. Target RNA shown as sticks. (D) Linear schematic of crystallized guide and target RNAs.

https://doi.org/10.7554/eLife.07646.003
The t1 nucleotide binding pocket specifically recognizes adenosine Strong electron density was observed for adenosine in the t1-binding pocket, but not t1C, G, or U.

(A) T1A target RNA omit map contoured at 3σ (grey mesh). (B) T1C target RNA omit map contoured at 3σ (grey mesh). (C) T1G target RNA omit map contoured at 3σ (grey mesh). (D) T1U target RNA omit map contoured at 3σ (grey mesh). (E) Plot of target bound vs Ago2-guide concentration for target RNAs with different t1-nucleotides.

https://doi.org/10.7554/eLife.07646.005
A purine N6 amine is required for t1 nucleotide recognition.

(A) Chemical structures of adenosine, 2,6-diaminopurine (DAP), guanosine, and inosine (I) nucelobases. Hydrogen bond donors highlighted in blue; hydrogen bond acceptors highlighted in pink. (B) t1I target RNA omit map contoured at 3σ (grey mesh). No electron density was observed for t1I. (C) t1DAP target RNA omit map contoured at 3σ (grey mesh). Clear electron density was observed for t1DAP. (D) Plot of target bound vs Ago2-guide concentration for t1A, t1I, t1DAP, and t1m6A target RNAs.

https://doi.org/10.7554/eLife.07646.006
Water-mediated recognition of t1A.

(A) Water network within the t1-binding pocket. Protein main chain shown as sticks, with side-chains (except S561) hidden for clarity. Water molecules shown as pink spheres. Water omit map shown contoured at 2.5σ (grey mesh). Potential hydrogen bonds shown as black dashed lines; t1A shown as blue sticks. (B) Flattened cartoon schematic of image shown in (A). Drawing illustrates proposed positions of hydrogen atoms within the t1A recognition network.

https://doi.org/10.7554/eLife.07646.008
Disruption of the t1 pocket water network abolishes t1A recognition.

(A) Overlay of wild-type and A481T Ago2 structures and close-up view of the region surrounding the A481T mutation (inset). (B, C) Crystal structures of A481T-Ago2 bound to t1A (B) or t1G (C) target RNAs. Target RNA omit maps contoured at 3σ (grey mesh). (D) Plot of bound t1A and t1G target RNAs vs A481T-Ago2-guide concentration. (E) Overlay of A481T-Ago2 and t1A from wild-type (WT) Ago2 (semi-transparent) structures. 4.2 Å distance between the T481 Oγ hydroxyl and the t1A N6 amine indicated as dashed line.

https://doi.org/10.7554/eLife.07646.010
t1A nucleotides increase the dwell time on target sites.

(A) Cartoon schematic of a single-molecule FRET assay. (B) Sequences of miRNA and target RNA with base pairs shown. The donor fluorophore (Cy3) is positioned on the ninth nt of miRNA (counting from the 5′ end of miRNA) and the acceptor (Cy5) on target RNA opposite nt 17 of miRNA. (C) Representative time trajectory. Δτ, dwell time of interaction; kon(obs), apparent binding rate. The thin grey box indicates the time of a flow that delivers Ago2 and microRNA into the observation chamber. (D) Accumulated number of first Ago2-miRNA/target binding events vs time for RNA targets bearing t1U, t1A, or t1DAP. The number of events was normalized by the total number of target RNA strands over an imaging area. (E) Binding event dwell times fit to a double exponential decay (blue). The t1A binding events fit two populations (49.8 ± 6.2% that exhibits Δτ1 and 50.2 ± 6.2% that exhibits Δτ2; R2 = 0.998). The t1DAP binding events fit two populations (44.6 ± 4.4% that exhibits Δτ1 and 55.4 ± 4.4% that exhibits Δτ2; R2 = 0.996). Dotted grey lines represent fits to a single exponential decay. The first data columns were excluded to avoid artifacts arising from the time resolution limit. The number of events per bin was normalized by the total amount of binding events per each histogram.

https://doi.org/10.7554/eLife.07646.011
Conservation in the t1A-binding pocket.

(A) Structure of the Ago2 t1A-binding pocket with major structural elements indicated. Ago2 shown as sticks with side chains of non-highlighted residues hidden for clarity. Target RNA shown in blue. (B) Multiple sequence alignment of Ago2 with other members of the extended Argonaute protein family. Conserved structural elements colored as in panel A.

https://doi.org/10.7554/eLife.07646.012
Author response image 1
Electron density maps surrounding the t1-G nucleotide.

2Fo-Fc map is shown contoured at 1σ(blue mesh). Fo-Fc map (difference map) is shown contoured at 3σ (green mesh) and -3σ (red mesh). A positive difference density peak, possibly representing a repositioned water C, is observed 2.5 Å from water B and 2.7 Å from the t1-G N2 amine. Position of water C in t1-A structure is included for comparison.

https://doi.org/10.7554/eLife.07646.015
Author response image 2
Photobleaching of the Cy3 donor.
https://doi.org/10.7554/eLife.07646.016

Tables

Table 1

Crystallographic and refinement statistics for wild-type Ago2-guide-target complexes

https://doi.org/10.7554/eLife.07646.004
Target RNAt1-Ct1-Gt1-Ut1-DAPt1-Inosine
PDB code4Z4C4Z4D4Z4E4Z4F4Z4G
Space groupP1211P1211P1211P1211P1211
Unit cell dimensions
a (Å)55.6955.7455.6455.8655.66
b (Å)116.56117.02116.84116.60117.0
c (Å)69.6169.8769.7470.3870.1
 β (°)92.4392.4392.4392.5292.40
Ago2 per ASU11111
Data collection
 Wavelength (Å)0.979450.979500.979180.979500.97950
 Resolution (Å)38.85–2.30 (2.38–2.30)39.01–1.60 (1.63–1.60)55.60–1.80 (1.90–1.80)38.86–2.80 (2.95–2.80)39.01–2.70 (2.83–2.70)
 Total reflections133,678532,622351,63465,58775,249
 Unique reflections38,61411377582,07121,07824,061
 Completeness (%)98.4 (96.3)96.8 (93.5)99.7 (99.7)95.0 (92.6)97.2 (91.9)
 Redundancy3.5 (3.4)4.7 (4.6)4.3 (3.7)3.1 (3.0)3.1 (2.9)
 I/σI13.1 (2.2)13.7 (2.0)10.3 (1.9)9.9 (2.4)9.6 (2.2)
 Rmerge7.7 (53.0)5.5 (74.9)9.8 (81.9)9.8 (57.7)8.4 (48.2)
 Rpim7.3 (49.2)3.1 (59.0)5.3 (47.4)9.7 (57.1)6.4 (36.3)
Refinement
 Resolution (Å)35.30–2.3039.01–1.6040.27–1.8035.47–2.8035.41–2.70
 R-free/R-factor21.86/16.9318.90/16.3118.54/15.7523.30/18.3922.44/17.86
 R.M.S. deviation
  Bond distances (Å)0.0140.0140.0070.0060.008
  Bond angles (°)1.4541.4601.1310.9030.989
 Number of atoms
  Non-hydrogen, protein64296469642964216404
  Non-hydrogen, RNA568580552571572
  Phenol2828282128
  Isopropanol420800
  Phosphate100500
  Mg33333
  Water27955454516105
 Ramachandran Plot
  Most favored regions95.33%96.79%96.97%94.93%95.17%
  Additionally allowed4.54%3.21%3.03%4.94%4.83%
  Generously llowed0.13%0.00%0.00%0.13%0.00%
Table 2

Ago2-target affinities

https://doi.org/10.7554/eLife.07646.007
t1 nucleotideKD (nM)
WT Ago2A481T-Ago2
A0.75 ± 0.041.5 ± 0.13
G1.9 ± 0.091.8 ± 0.11
U1.9 ± 0.10
C1.8 ± 0.12
DAP0.45 ± 0.03
I1.7 ± 0.09
m6A1.8 ± 0.12
  1. Dissociation constants for wild-type (WT) and mutant Ago2 binding short target RNAs with different t1 nucleotides.

Table 3

Crystallographic and refinement statistics for mutant (A481T) Ago2-guide-target complexes

https://doi.org/10.7554/eLife.07646.009
Target RNAt1-At1-G
PDB code4Z4H4Z4I
Space groupP1211P1211
Unit cell dimensions
a (Å)55.6955.60
b (Å)116.60116.60
c (Å)70.1069.62
 β (°)92.2992.42
Ago2 per ASU11
Data collection
 Wavelength (Å)0.999990.99999
 Resolution (Å)44.81–2.50 (2.61–2.50)44.69–2.80 (2.95–2.80)
 Total reflections11333285,923
 Unique reflections30,41121,701
 Completeness (%)98.6 (98.3)99.2 (97.6)
 Redundancy3.7 (3.8)4.0 (4.0)
 I/σI9.2 (2.3)8.7 (2.4)
 Rmerge12.7 (69.7)13.1 (60.0)
 Rpim12.0 (65.0)12.0 (55.0)
Refinement
 Resolution (Å)44.81–2.5044.69–2.80
 R-free/R-factor21.13/17.2123.32/19.18
 R.M.S. deviation
  Bond distances (Å)0.0080.005
  Bond angles (°)0.9400.865
 Number of atoms
  Non-hydrogen, protein64326412
  Non-hydrogen, RNA568568
  Phenol2821
  Isopropanol420
  Phosphate100
  Mg33
  Water13523
 Ramachandran plot
  Most favored regions95.19%94.67%
  Additionally allowed4.68%5.33%
  Generously allowed0.13%0.00%

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