www.sciencemag.org/cgi/content/full/science.1221551/DC1
Supplementary Materials for
The Crystal Structure of Human Argonaute2
Nicole T. Schirle and Ian J. MacRae*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 27 April 2012 on Science Express
DOI: 10.1126/science.1221551
This PDF file includes:
Materials and Methods
Figs. S1 to S15
Tables S1 and S2
Full References
Methods and Materials
Native Protein Expression and Purification
The full-length human Ago2 cDNA (NP_036286) was cloned into pFastBac HT A
for the production of Ago2-expressing baculovirus using the bac-2-bac system
(Invitrogen) as described previously (32). Point mutations were introduced into the Ago2
gene using QuickChange mutatgenesis (Stragegene). Wild-type and mutant Ago2
proteins were expressed in Sf-9 cells with an N-terminal hexa-histidine tag upstream of
a tobacco etch virus (TEV) protease cleavage site. Ago2 was purified from clarified
lysate using standard nickel-affinity purification with an agarose Ni-NTA column and
cellular RNAs were degraded by extensive on-resin treatment with RNase A. The Histag was removed using TEV protease after elution from the Ni-NTA resin. The protein
was then passed over a second Ni-NTA column and the unbound protein was collected
and further purified using size exclusion chromatography. Purified Ago2 was
concentrated to 3 mg/mL in 10 mM Tris (pH 8.0), 0.1 M NaCl, and 0.5 mM Tris(2carboxyethyl) phosphine hydrochloride (TCEP) and stored at -80 ˚C.
Selenomethionine-derivatized (SeMet) Protein Expression and Purification
Sf-9 cells were infected with baculovirus expressing full-length human Ago2 in
standard growth media (ESF 921, Expression Systems, Woodland CA) for 8 hours.
Cells were harvested and re-suspended in methionine-deficient medium and grown for
another 8 hours. SeMet was then added to the growth medium to a final concentration
of 100 mg/L. Cells were harvested for protein purification 48 hours after the addition of
SeMet. SeMet Ago2 was purified and crystallized using the same purification protocol
as for native protein.
Crystallization and Data Collection
Crystals were grown at 20°C using hanging drop vapor diffusion and usually
appeared within 24 hours. Drops contained a 1.0:0.8 ratio of protein (3 mg/mL) and
reservoir solution (16% PEG 3350, 0.1 M phenol, 12% isopropanol, and 0.1 M Tris (pH
9.0)). Crystals were harvested with nylon loops (no cat whiskers required, sorry
Sproket) and soaked in reservoir solution containing 25% ethylene glycol as cryoprotectant before cryo-cooling by plunging into liquid N2. We found that crystals grown
from mutant forms of Ago2, in which serine residues that were phosphorylated in the
Sf9 cells were mutated to either alanine or aspartate, tended to diffract x-rays more
strongly than crystals of the wild-type protein. The best diffracting construct, which was
used for both reported structures had five point mutations: S387D, S824A, S828D,
S831D, and S834A. Native data were collected under cryo-conditions on Beamline 24ID-E at the Advanced Photon Source (APS). SeMet diffraction data were collected on
beamline 11-1 at the Stanford Synchrotron Radiation Lightsource (SSRL). Native data
were processed using HKL2000 (33) and SeMet data were processed using XDS and
scala (34).
Ago2-tryptophan crystals were grown using hanging drop vapor diffusion at 20°C
and appeared within 48 hours. Drops contained a 1.0:0.8 ratio of protein (3 mg/mL) to
reservoir solution (16% PEG 3350, 12% isopropanol, L-tryptophan and 0.1M Tris (pH
9.0)). Crystals were harvested for x-ray data collection by soaking first in reservoir
solution containing 12% ethylene glycol, followed by further soaking in reservoir solution
containing 25% ethylene glycol as cryo-protectant. Following cryo-protection, crystals
were cryo-cooled by plunging into liquid N2. Data were collected on beamline 11-1 at
the Stanford Synchrotron Radiation Lightsource. Data were processed using XDS and
scala (34). All indexed crystals belonged to the monoclinic space group P21 with one
Ago2 in the asymmetric unit.
Structure Determination
Nineteen Selenium sites were identified using the HySS routine in PHENIX (35).
These sites were used to calculate an initial map using SOLVE followed by solvent
flattening with RESOLVE (36). An initial model was built into the solvent flattened map
using Coot (37). The initial model was subjected to real space refinement of XYZ
coordinates, and individual b-factors (and occupancy for nucleotides 8, 9 and 21 of the
RNA) against the native data using PHENIX. Several iterative rounds of model building
and refinement (using PHENIX or Refmac (38)) were performed until all interpretable
electron density was modeled. Water molecules were identified automatically in Coot
(2Fobs – Fcalc peaks, 1.8σ or higher, that were greater than 2.4 Å and less than 3.2 Å
from a hydrogen bond donor or acceptor) and by manual inspection of electron density
maps. Default target bond and angle values for RNA chains were used in all
refinements. The native model was refined against the Ago2-tryptophan data set using
Refmac (38). Tryptophan molecules were identified by manual inspection of 2Fobs – Fcalc
difference maps. All structures used the same test data set for calculating Rfree values.
Analysis of Ago2-Bound RNA
RNA associated with purified human Ago2 was removed from the protein by
phenol-chloroform extraction and subsequent ethanol precipitation. Isolated RNA was
treated with calf-intestinal phosphatase followed by another phenol-chloroform
extraction and ethanol precipitation. Dephosphorylated RNA was 5'-end labeled by
treating with γ-32P-ATP and polynucleotide kinase. Synthetic 20mer and 10mer RNAs
were
32
P-labeled for size comparison. Bound guide RNA fragments were resolved by
denaturing polyacrylamide (14%) gel electrophoresis and visualized by phosphor
imaging.
Figure S1. Superposition of Ago2 onto prokaryotic Argonautes. Aligning the PIWI domain
of Ago2 to three prokaryotic Argonaute structures reveals that the overall structure of human
Ago2 resembles its prokaryotic homologs. (A) Alignment of T. thermophilus Argonaute in an
“open” conformation (PDB ID: 3F73) with human Ago2. Human Ago2 and T. thermophilus Ago
are shown in yellow and pink, respectively. (B) Alignment of T. thermophilus Ago in a “closed”
conformation (PDB ID: 3DLH) with human Ago2. T. thermophilus Ago shown in green. (C)
Alignment of P. furiosus Ago (PDB ID: 1U04) with human Ago2. P. furiosus Ago is shown in
blue. An arrow indicates the location of α-helix-7 in human Ago2. Human Ago2 appears to have
crystallized in a conformation most similar to the “open” state observed in the T. thermophilus
Ago, which is bound to a guide DNA and target RNA duplex. The Apo P. furiosus Ago, observed
in a “closed” conformation relative to human Ago2, appears to have an α-helix homologous to
α-helix-7 in human Ago2.
Figure S2. Orientation of the MID-PIWI interface varies between Kingdoms of Life.
Alignment of the PIWI domains of T. thermophilus Ago bound to a DNA-RNA duplex (A), T.
thermophilus Ago bound to a DNA guide (B), P. furiosus Ago (C), and N. crassa QDE-2 (D) with
Ago2. The complete structure of Ago2 is shown in yellow. The MID-PIWI lobe of: T.t. Ago bound
to a DNA-RNA duplex is shown in pink (PDB ID: 3F73); T.t. Ago bound to a guide DNA is shown
in green (PDB ID: 3DLH); P.f. Ago is shown in blue (PDB ID: 1U04); and N.c. QDE-2 is shown
in grey (rmsd 1.61 Å, 172 Cα, PDB ID: 2YHA).
The MID domain of Ago2 is shifted relative to its PIWI domain when compared to the MID-PIWI
domains for Argonautes from bacteria and archaea. In contrast, the MID-PIWI lobe of Ago2 and
N. crassa QDE-2 superimpose well, revealing that the domain arrangement is conserved
between distant members of eukarya.
Figure S3. Alignment of Argonaute Sequences. Sequence alignment of human Ago2
(GenBank 29171734), human Ago1 (GenBank 6912352), Drosophila Ago1 (GenBank
24653501), and N. crassa QDE-2 (GenBank 74625571). Alpha helices and beta strands are
indicated by green loops or blue arrows, respectively. Residues that contribute to the kink in the
guide RNA are shown in pink. Residues that make contacts to guide RNA phosphates are
shown in orange. Catalytic residues in the active site are shown in red. Grey letters indicate
disordered loops not observed in the Ago2 crystal structure
Figure S4. Structure-based alignment of Ago2 and T. thermophilus Ago sequences. Ago2
(GenBank 29171734) and T. thermophilus Ago (Genbank 46255097) sequences are aligned.
An initial sequence alignment from ClustalW was manually edited to best align secondary
structural elements in human Ago2 and TtAgo crystal structures. Alpha helices and beta strands
are indicated by green loops or blue arrows, respectively. Eukaryotic specific insertions are
shown in orange and catalytic residues in red.
Figure S5. Location of eukaryotic-specific insertions. The core domains in Ago2 have
extended loops and additional secondary structures (highlighted in orange) that are not present
in T. thermophilus. Several notable extensions surround the region associated with binding to
nucleotides in the 5' half of the guide. These include a large eukaryotic-specific insertion in the
PIWI domain that is disordered in our structure. This insertion was suggested to bind to target
RNAs and extend across the nucleic acid binding cleft to interact with the PAZ domain, forming
a clamp around bound guide-target duplexes (1). Consistent with this notion the insertion is
positioned across the nucleic acid binding cleft from a eukaryotic-specific insertion (helix-5) in
the PAZ domain. Guide RNA is shown in violet. Tryptophan molecules are colored blue.
Figure S6. Eukaryotic insertion in the PIWI domain may interact with PAZ.
The eukaryotic insertion observed in the N. crassa QDE-2 PIWI domain crystal structure was
disordered in human Ago2. However, superimposing QDE-2 (blue – PDB ID: 2YHA) onto Ago2
suggests that the eukaryotic insertion could interact with a helix-5 (α5) in the human Ago2 PAZ
domain. Human Ago2 is colored grey except for these two eukaryotic-specific regions, which
are colored orange.
Figure S7. RNA bound to Ago2 is a heterogeneous mixture of RNAs approximately 10-20
nucleotides in length. Synthetic 10- and 20-mer RNAs were labeled as size markers.
Figure S8. A guide RNA is bound to human Ago2. (A) Experimental electron density map
after solvent flattening, contoured at 1 sigma, surrounding nucleotides 1-9 of the guide RNA
bound to human Ago2. RNA bases, are shown in red as sticks. (B) Refined 22Fobs – Fcalc map
of guide RNA nucleotides 1-9. Most 2' hydroxyls are unambiguously observed in the refined
map. (C) Superposition of the human Ago2 guide RNA with an A-form dsRNA (blue, derived
from PDB ID 3CIY). A kink introduced by human Ago2 between bases 6 and 7 of the guide RNA
breaks the A-form structure of the guide, potentially disrupting base pairing to complementary
target RNAs.
Figure S9. Contacts to the guide RNA 2' hydroxyls. Detailed interactions between Ago2 and
the guide RNA are shown. 2' hydroxyls are numbered. Potential hydrogen bonds to the guide
RNA 2' hydroxyls are indicated as dashed orange lines. Ago2 domains are colored as in Fig. 1.
Water molecules are shown as pink spheres.
Figure S10. Electrostatic surfaces of T. thermophilus and human Argonautes. (A)
Electrostatic surface representation of human Ago2. Guide RNA is shown in yellow as sticks.
(B) Close up view of the guide RNA 5’-binding pocket in human Ago2. Parts of the PAZ domain
where removed for clearer viewing. (C) Electrostatic surface representation of TtAgo (PDB ID:
3DLH). Guide DNA is shown in yellow as sticks. (D) Close up of the guide DNA 5’-binding
pocket in TtAgo. Parts of the PAZ domain were removed. The guide-binding surface is more
hydrophobic in TtAgo than in Ago2.
Figure S11. Contacts made by helix-7. The observed kink between nucleotides 6 and 7 of the
guide RNA appears to be stabilized by helix-7 in the L2 domain of Ago2. Helix-7 is physically
inserted between the nucleotide bases 6 and 7. Although we cannot formally exclude the
possibility that the observed position of helix-7 is an artifact of crystallization, inspection of
crystal packing in this region suggests this is not the case. Helix-7 makes a single interaction
with an adjacent molecule in the crystal lattice (a suboptimally aligned hydrogen bond between
N359 of L2 and H81 of the adjacent molecule). In contrast, helix-7 makes extensive
intermolecular contacts with the guide RNA and the L1 domain. These observations suggest
that the crystallized state is a conformation of Ago2 that is naturally explored by the enzyme in
free solution. We hypothesize this conformation may be associated with release of passenger
strands or sliced targets.
Figure S12. Docked target RNAs clash with helix-7. (A) Close up view of the Ago2 guide
RNA (red) base paired to a docked A-form target RNA (blue). Guide RNA nucleotides are
numbered from the 5' end. Target nucleotides are numbered as paired to the guide. The target
is well accommodated within Ago2 except for a steric clash with helix-7 (α7). (B) Opposite view
of that show in (A). Sections of the MID and PIWI domains were removed for clarity.
Figure S13. Phenol in tryptophan binding site 1. (A) Tryptophan bound in site 1 of the PIWI
domain is shown. 2Fobs – Fcalc map (calculated before tryptophan was included in the model)
contoured at 2.5 sigma, surrounding the tryptophan is shown as an orange wire mesh. (B) A
single phenol molecule bound to site 1 in the absence of tryptophan. Refined 2Fobs – Fcalc omit
map, contoured at 2.5 sigma, surrounding the phenol is shown as a green wire mesh. (C)
Superimposing tryptophan and phenol bound structures reveals that the two small molecules
bind in almost exactly the same position.
Figure S14. Mutations known to disrupt GW182 binding to Argonaute. Mutagenesis studies
from several independent groups have identified amino acid residues in human Ago2 and
Drosophila Ago1 that are required for Argonaute to bind to GW182 (25, 29-31). These studies
consistently found that mutations that disrupt miRNA binding to Argonaute also block the
binding of GW182. However, the converse is not true—some mutations were identified in
Argonaute that disrupt the association with GW182 without affecting miRNA binding. These
observations suggest that GW182 binding occurs downstream of miRNA loading. (A) Amino
acid residues, identified by mutagenesis, required for binding GW182 are shown as sticks on
the structure of Ago2. Most mutations cluster around two regions of the protein: the binding site
for the 5' end of guide RNAs, and the tryptophan binding sites. Residues colored red and violet
are required for binding both miRNAs and GW182. Residues colored green are required for
binding GW182 only (miRNA binding unaffected). All residues that are specifically required for
GW182 binding cluster around the tryptophan binding pockets. (B) Close up view of the
residues required for GW182 interactions near the tryptophan binding sites. Side chains are
numbered according to the human Ago2 sequence with corresponding the Drosophila Ago1
residues numbered in parentheses. (C) Close up view of the F2V2 mutation sites. These
phenylalanines contribute to the hydrophobic core of the MID domain. Disruption of these
interactions would likely cause local misfolding of the MID domain. (D) Close up view the of the
5’-nucleotide binding site with mutation sites known to disrupt miRNA and GW182 binding
shown.
Mutation sites tested for GW182 binding in both human Ago2 and Drosophila Ago1 are as
follows: F470, F505, Y529, K533, R583, I592, Y625, R647, F653, and K660 (25, 29-31). Sites
tested only in human Ago2 are: Q545, and R812 (29). Equivalent sites tested only in Drosophila
Ago1 are: K566, and K570 (25, 30, 31).
Figure S15. The Active Site of Human Ago2. Superposition of human Ago2 with T.
thermophilus Ago (TtAgo) (PDB ID: 3HVR) and P. furiosus (PfAgo) (PDB ID: 1U04) near the
PIWI domain active site indicates that the active site of human Ago2 forms a cleavagecompetent RNA-binding pocket. Human Ago2, TtAgo, and PfAgo are shown in yellow, violet,
and blue, respectively. Magnesium ions from the TtAgo structure are shown as green spheres.
Table S1. Crystallographic Statistics – Native and Heavy Atom Data
Space group
Unit Cell Dimensions
a, b, c (Å)
α, β, γ (°)
P1211
63.1, 107.6, 68.5
90.0, 107.1, 90.0
Ago2 molecules per ASU 1
Data Collection
Native
Wavelength (Å)
Resolution (Å)
No. Reflections
Total
Unique
Completeness (%)
Redundancy
I/σI
Rmerge (%)
No. Sites
Figure of Merit
Se (peak)
Se (remote) 0.97920
0.97926
50-2.28 (2.37-2.28) 39-3.0 (3.1-3.0)
0.91837 39-3.0 (3.1-3.0)
421625
44672
99.4 (98.9)
3.8 (3.8)
5.5 (2.4)
5.7 (55.1)
241434
17422
98.8 (96.9)
13.8 (13.5)
11.6 (3.0)
5.9 (25.7)
Refinement
Resolution (Å)
R-free/R-factor
R.M.S. Deviation
Bond Distances (Å)
Bond Angles (°)
Number of Atoms
Non-hydrogen, protein
Non-hydrogen, RNA
Phenol
Isopropanol
Water
Ramachandran Plot
Most Favored Regions
Additionally Allowed
Generously Allowed
241512
17413
98.9 (97.7)
13.8 (13.9)
12.0 (3.5)
5.5 (21.8)
19
0.698
65.4-2.3
26.95/21.12
0.014
1.577
6335
191
7
4
99
94.81%
4.68%
0.52%
Table S2. Crystallographic Statistics – Tryptophan Data
Space group
Unit Cell Dimensions
a, b, c (Å)
α, β, γ (°)
P1211
63.2, 106.7, 68.3
90.0, 107.0, 90.0
Ago2 molecules per ASU 1
Data Collection
Wavelength (Å)
Resolution
No. Reflections
Total
Unique
Completeness (%)
Redundancy
I/σI
Rmerge (%)
Tryptophan
0.97945
39-2.9 (3.0-2.9)
65312
19020
97.9 (93.2)
3.4 (3.2)
10.8 (2.6)
5.7 (28.6)
Refinement
Resolution
R-free/R-factor
R.M.S. Deviation
Bond Distances (Å)
Bond Angles (°)
Number of Atoms
Non-hydrogen, protein
Non-hydrogen, RNA
Tryptophans
Water
Ramachandran Plot
Most Favored Regions
Additionally Allowed
Generously Allowed
65.4-2.90
24.49/19.51
0.011
1.556
6372
191
28
15
95.35%
4.26%
0.39%
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