Crystal Structure of Argonaute
and Its Implications for RISC
Slicer Activity
Ji-Joon Song, Stephanie K. Smith, Gregory J. Hannon,
Leemor Joshua-Tor
Pamela Lussier
Biochemistry 4000/5000
http://www.nature.com/f
ocus/rnai/animations/ani
mation/animation.htm
RNA interference (RNAi)
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Triggered by the presence of dsRNA
RNase III family enzyme Dicer
initiates silencing by releasing siRNA ~ 20
base duplexes with two-nucleotide 3’
overhangs
siRNA guide substrate selection by effector
complexes called RISC
RISC
(RNA induced Silencing Complex)
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Contain single stranded versions of siRNA
as well as additional protein components
One of which is a member of the
Argonaute family of proteins
RISC recognizes and destroys target
mRNAs by cleavage in region homologous
to siRNA.
Argonaute Protein
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Defined by presence of PAZ (Piwi
Argonaute Zwille) and PIWI (named for
protein piwi) domains
PAZ domain of Argonaute interacts directly
with small RNA in RISC
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Forms a oligonucleotide/oligosaccharide
binding (OB) fold containing a central cleft
lined with conserved aromatic residues that
bind specifically to single stranded 3’ ends
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In RISC, the Argonaute PAZ domain would
hold the 3’ end of the single stranded
siRNA
Possibly orients recognition and cleavage
of mRNA substrates.
Nuclease Responsible for cleavage (Slicer)
has escaped identification.
Objective
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Deepen understanding of the role of
Argonaute protein in RNAi
To conduct structural studies of a full
length Argonaute protein from Pyrococcus
furiosus.
Hanging Drop Method
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Initial crystals
were grown by
vapor diffusion
using hangingdrop method in
presence of small
amounts of
organic solvent
Quality of crystals improved by
microseeding
Control nucleation
as it consists of
introducing crystal
nuclei in the
equilibrated
metastable protein
solution, where
seeds might grow
Structural Determination
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Multiple Anomalous Dispersion (MAD)
Used selenomethionine substituted protein
crystal.
Structure of full-length Argonaute
(PfAgo) determined to 2.25 Angstroms.
> 90%
Good is <20% (0.2)
Crystal Structure of P. furiosus
Argonaute
Overall architecture
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N-terminal, middle, and PIWI domains form a
crescent-shaped base
N-terminal domain forms a “stalk” that holds
PAZ domain above the crescent and an
interdomain connector cradles the four domains
of the molecule.
Forms a groove at the center of the crescent
and the PAZ domain closes off the top of this
groove.
PAZ Domain
Red = PfAgo
Gray = hAgo1
Dotted lines in figure
represent disordered regions
Sequence Alignment of PAZ domains of
PfAgo, hAgo1 and DmAgo2
Primary sequence comparisons failed to reveal PAZ
domain despite close structural similarities
Purple = invariant residues
Blue = conserved residues
PAZ domain
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Conserved aromatic
residues that bind the
two-nucleotide 3’
overhang of an siRNA
are present in PfAgo.
Side chains occupy
similar positions in
space, but they are
anchored to peptide
backbone in different
locations.
Green = hAgo1 residues
So…where does the 3’ overhang of
the siRNA bind???
Right Here!!!
PAZ Domain Comparison
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L263 and I261 assume role of L337 and T335 in
hAgo1, which anchor sugar ring of terminal
residue through vand der Waals interactions.
W213 assume role of F292 in hAgo1, which stacks
against the terminal nucleotide.
R220 is positioned similarly to K313 that contacts
the penultimate nucleotide.
Reasoned that the PAZ domain in PfAgo
binds RNA 3’ ends, as do PAZ domains
of fly and human Argonautes.
PIWI is an RNase H Domain
The domains are topologically identical
Five-stranded mixed β sheet surrounded by helices
PIWI Domain
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3 highly conserved catalytic carboxylates
One is located in β1, and one is located at
C terminus of fourth strand β4
The third carboxylate varies
Only requirement is a reasonable spatial
position at the active site.
Active Site Rotated 180°
Two aspartate residues in PIWI were located at same positions as
the invariant carboxylates – D558 on first β strand, and D628 on
the end of the fourth strand.
E635 is in close proximity to the two aspartates and suggests
that this glutamate serves as the third active-site residue.
Active site is positioned in a cleft in the
middle of the crescent in the groove
below the PAZ domain.
Here on overall
structure
Ago is Slicer
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Argonaute is the enzyme in RISC that
cleaves the mRNA.
RNase H enzymes cleave ssRNA “guided”
by DNA strand in RNA/DNA hybrid.
Argonautes might do RNA cleavage guided
by the siRNA strand in a dsRNA substrate.
Depends on Mg+2, like other RNase H
enzymes.
Distinct groove
throughout the protein,
which has a claw shape
and bends between the
PAZ and N-terminal
domains.
Blue = positively charged
Approx location of active site
marked by a yellow asterisk
Electrostatics show
inner groove is lined
with positive charges –
suitable for interaction
with negatively charged
phosphate backbone.
Possible substrate binding
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Superimposed PAZ
domains from PfAgo
and hAgo1 and
examined position of
RNA in hAgo1
complex with respect
to PfAgo.
siRNA guide interacts
with PAZ cleft
Model for siRNA-guided mRNA
cleavage
siRNA binds with its 3’ end in the
PAZ cleft and the 5’ is predicted
to bind near the other end of
the cleft.
The mRNA comes in between
the N-terminal and PAZ domains
and out between the PAZ and
middle domain.
The active site in the PIWI
domain cleaves the mRNA
opposite the middle of the siRNA
guide.
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From studies of other RNase H enzymes,
expected that Argonaute senses the minor
groove width of the dsRNA, which differs
from that of dsDNA.
Fits with RISC’s inability to cut DNA
substrates.
Opening of the claw might assist binding
of mRNA – hinge region exists in
interdomain connector at residues 317320.
Added Support
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In mammalian system, performed
mutational analysis on hAgo2
Conserved active site aspartates were
altered = loss of nuclease activity but
retained siRNA binding.
Remaining Questions?
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Other determinants beyond catalytic triad
of PIWI domain that determine activity
toward RNA substrates, such as
conformational differences.
Interactions with other factors may be
needed to create fully active Slicer
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Identification of catalytic center of RISC
awaited a drive towards understanding
RNAi at a structural level.
A full understanding of the underlying
mechanism of RNAi will need to be
derived from a combination of biochemical
and structural studies of RISC.