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 (RNAi) Triggered by the presence of dsRNA Triggered by the presence of dsRNA RNase III family enzyme Dicer RNase III family enzyme Dicer initiates silencing by releasing siRNA ~ 20 base duplexes with two-nucleotide 3’ overhangs initiates silencing by releasing siRNA ~ 20 base duplexes with two-nucleotide 3’ overhangs siRNA guide substrate selection by effector complexes called RISC siRNA guide substrate selection by effector complexes called RISC

RISC (RNA induced Silencing Complex) Contain single stranded versions of siRNA as well as additional protein components Contain single stranded versions of siRNA as well as additional protein components One of which is a member of the Argonaute family of proteins 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. RISC recognizes and destroys target mRNAs by cleavage in region homologous to siRNA.

Argonaute Protein Defined by presence of PAZ (Piwi Argonaute Zwille) and PIWI (named for protein piwi) domains 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 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 Forms a oligonucleotide/oligosaccharide binding (OB) fold containing a central cleft lined with conserved aromatic residues that bind specifically to single stranded 3’ ends

In RISC, the Argonaute PAZ domain would hold the 3’ end of the single stranded siRNA In RISC, the Argonaute PAZ domain would hold the 3’ end of the single stranded siRNA Possibly orients recognition and cleavage of mRNA substrates. Possibly orients recognition and cleavage of mRNA substrates. Nuclease Responsible for cleavage (Slicer) has escaped identification. Nuclease Responsible for cleavage (Slicer) has escaped identification.

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

Hanging Drop Method Initial crystals were grown by vapor diffusion using hanging- drop method in presence of small amounts of organic solvent Initial crystals were grown by vapor diffusion using hanging- drop 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 Multiple Anomalous Dispersion (MAD) Multiple Anomalous Dispersion (MAD) Used selenomethionine substituted protein crystal. Used selenomethionine substituted protein crystal. Structure of full-length Argonaute (PfAgo) determined to 2.25 Angstroms. 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 N-terminal, middle, and PIWI domains form a crescent-shaped base 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. 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. 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 Conserved aromatic residues that bind the two-nucleotide 3’ overhang of an siRNA are present in PfAgo. 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. 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 L 263 and I 261 assume role of L 337 and T 335 in hAgo1, which anchor sugar ring of terminal residue through vand der Waals interactions. L 263 and I 261 assume role of L 337 and T 335 in hAgo1, which anchor sugar ring of terminal residue through vand der Waals interactions. W 213 assume role of F 292 in hAgo1, which stacks against the terminal nucleotide. W 213 assume role of F 292 in hAgo1, which stacks against the terminal nucleotide. R 220 is positioned similarly to K 313 that contacts the penultimate nucleotide. R 220 is positioned similarly to K 313 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. 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 3 highly conserved catalytic carboxylates 3 highly conserved catalytic carboxylates One is located in β1, and one is located at C terminus of fourth strand β4 One is located in β1, and one is located at C terminus of fourth strand β4 The third carboxylate varies The third carboxylate varies Only requirement is a reasonable spatial position at the active site. 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 – D 558 on first β strand, and D 628 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 Argonaute is the enzyme in RISC that cleaves the mRNA. Argonaute is the enzyme in RISC that cleaves the mRNA. RNase H enzymes cleave ssRNA “guided” by DNA strand in RNA/DNA hybrid. 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. Argonautes might do RNA cleavage guided by the siRNA strand in a dsRNA substrate. Depends on Mg +2, like other RNase H enzymes. 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. 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

Possible substrate binding Superimposed PAZ domains from PfAgo and hAgo1 and examined position of RNA in hAgo1 complex with respect to PfAgo. 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 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.

From studies of other RNase H enzymes, expected that Argonaute senses the minor groove width of the dsRNA, which differs from that of dsDNA. 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. 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 Opening of the claw might assist binding of mRNA – hinge region exists in interdomain connector at residues

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

Remaining Questions? Other determinants beyond catalytic triad of PIWI domain that determine activity toward RNA substrates, such as conformational differences. 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 Interactions with other factors may be needed to create fully active Slicer

Identification of catalytic center of RISC awaited a drive towards understanding RNAi at a structural level. 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. A full understanding of the underlying mechanism of RNAi will need to be derived from a combination of biochemical and structural studies of RISC.