Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity

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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
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RNA interference
http//www.nature.com/focus/rnai/animations/animat
ion/animation.htm
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RNA interference (RNAi)

• 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

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RISC (RNA induced Silencing Complex)

• 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.

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Argonaute Protein

• 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
• 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.

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Objective

• Deepen understanding of the role of Argonaute
  protein in RNAi
• To conduct structural studies of a full length
  Argonaute protein from Pyrococcus furiosus.

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Hanging Drop Method

• Initial crystals were grown by vapor diffusion
  using hanging-drop method in presence of small
  amounts of organic solvent

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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
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Structural Determination

• Multiple Anomalous Dispersion (MAD)
• Used selenomethionine substituted protein
  crystal.
• Structure of full-length Argonaute (PfAgo)
  determined to 2.25 Angstroms.

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gt 90
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Good is lt20 (0.2)
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Crystal Structure of P. furiosus Argonaute
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Overall architecture

• 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.

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PAZ Domain
Red PfAgo Gray hAgo1
Dotted lines in figure represent disordered
regions
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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
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PAZ domain

• 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
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Sowhere does the 3 overhang of the siRNA bind???
Right Here!!!
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PAZ Domain Comparison

• 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.

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PIWI is an RNase H Domain
The domains are topologically identical Five-stran
ded mixed ß sheet surrounded by helices
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(No Transcript)
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PIWI Domain

• 3 highly conserved catalytic carboxylates
• One is located in ß1, and one is located at C
  terminus of fourth strand ß4
• The third carboxylate varies
• Only requirement is a reasonable spatial position
  at the active site.

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Active Site Rotated 180
Two aspartate residues in PIWI were located at
same positions as the invariant carboxylates
D558 on first ß 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.
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Active site is positioned in a cleft in the
middle of the crescent in the groove below the
PAZ domain.
Here on overall structure
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Ago is Slicer

• 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 Mg2, like other RNase H enzymes.

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Distinct groove throughout the protein, which has
a claw shape and bends between the PAZ and
N-terminal domains. Electrostatics show inner
groove is lined with positive charges suitable
for interaction with negatively charged phosphate
backbone.
Blue positively charged Approx location of
active site marked by a yellow asterisk
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Possible substrate binding

• 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

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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 RISCs inability to cut DNA substrates.
• Opening of the claw might assist binding of mRNA
  hinge region exists in interdomain connector at
  residues 317-320.

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Added Support

• In mammalian system, performed mutational
  analysis on hAgo2
• Conserved active site aspartates were altered
  loss of nuclease activity but retained siRNA
  binding.

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Remaining Questions?

• 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.

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Questions?