Crystal Structure of Crystal Structure of Argonaute and Its Argonaute and Its

Implications for RISC Slicer Implications for RISC Slicer ActivityActivity

Ji-Joon Song, Stephanie K. Smith, Gregory J. Ji-Joon Song, Stephanie K. Smith, Gregory J. Hannon, Leemor Joshua-TorHannon, Leemor Joshua-Tor

Pamela LussierPamela Lussier

Biochemistry 4000/5000Biochemistry 4000/5000

http://www.nature.com/focus/rnai/animations/animation/animation.htm

RNA interference (RNAi)RNA interference (RNAi)

Triggered by the presence of dsRNATriggered by the presence of dsRNA RNase III family enzyme RNase III family enzyme DicerDicer initiates silencing by releasing siRNA initiates silencing by releasing siRNA

~ 20 base duplexes with two-~ 20 base duplexes with two-nucleotide 3’ overhangsnucleotide 3’ overhangs

siRNA guide substrate selection by siRNA guide substrate selection by effector complexes called RISCeffector complexes called RISC

RISC RISC (RNA induced Silencing Complex)(RNA induced Silencing Complex)

Contain single stranded versions of Contain single stranded versions of siRNA as well as additional protein siRNA as well as additional protein componentscomponents

One of which is a member of the One of which is a member of the Argonaute Argonaute family of proteinsfamily of proteins

RISC recognizes and destroys target RISC recognizes and destroys target mRNAs by cleavage in region mRNAs by cleavage in region homologous to siRNA.homologous to siRNA.

Argonaute ProteinArgonaute Protein

Defined by presence of PAZ (Piwi Defined by presence of PAZ (Piwi Argonaute Zwille) and PIWI (named for Argonaute Zwille) and PIWI (named for protein piwi) domainsprotein piwi) domains

PAZ domain of Argonaute interacts PAZ domain of Argonaute interacts directly with small RNA in RISCdirectly with small RNA in RISC Forms a oligonucleotide/oligosaccharide Forms a oligonucleotide/oligosaccharide

binding (OB) fold containing a central cleft binding (OB) fold containing a central cleft lined with conserved aromatic residues lined with conserved aromatic residues that bind specifically to single stranded 3’ that bind specifically to single stranded 3’ endsends

In RISC, the Argonaute PAZ domain In RISC, the Argonaute PAZ domain would hold the 3’ end of the single would hold the 3’ end of the single stranded siRNAstranded siRNA

Possibly orients recognition and Possibly orients recognition and cleavage of mRNA substrates.cleavage of mRNA substrates.

Nuclease Responsible for cleavage Nuclease Responsible for cleavage (Slicer) has escaped identification.(Slicer) has escaped identification.

ObjectiveObjective

Deepen understanding of the role of Deepen understanding of the role of Argonaute protein in RNAiArgonaute protein in RNAi

To conduct structural studies of a full To conduct structural studies of a full length Argonaute protein from length Argonaute protein from Pyrococcus furiosusPyrococcus furiosus..

Hanging Drop MethodHanging Drop Method

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

Quality of crystals improved by Quality of crystals improved by microseedingmicroseeding

Control nucleation as it consists of introducing crystal nuclei in the equilibrated metastable protein solution, where seeds might grow

Structural DeterminationStructural Determination

Multiple Anomalous Dispersion (MAD)Multiple Anomalous Dispersion (MAD) Used selenomethionine substituted Used selenomethionine substituted

protein crystal.protein crystal. Structure of Structure of full-lengthfull-length Argonaute Argonaute

(PfAgo) determined to 2.25 (PfAgo) determined to 2.25 Angstroms.Angstroms.

> 90%

Good is <20% (0.2)

Crystal Structure of Crystal Structure of P. furiosusP. furiosus ArgonauteArgonaute

Overall architectureOverall architecture

N-terminal, middle, and PIWI domains form N-terminal, middle, and PIWI domains form a crescent-shaped basea crescent-shaped base

N-terminal domain forms a “stalk” that N-terminal domain forms a “stalk” that holds PAZ domain above the crescent and holds PAZ domain above the crescent and an interdomain connector cradles the four an interdomain connector cradles the four domains of the molecule.domains of the molecule.

Forms a groove at the center of the Forms a groove at the center of the crescent and the PAZ domain closes off crescent and the PAZ domain closes off the top of this groove.the top of this groove.

PAZ DomainPAZ Domain

Red = PfAgo

Gray = hAgo1

Dotted lines in figure represent disordered regions

Sequence Alignment of PAZ Sequence Alignment of PAZ domains of PfAgo, hAgo1 and domains of PfAgo, hAgo1 and

DmAgo2DmAgo2

Primary sequence comparisons failed to reveal PAZ domain despite close structural similarities

Purple = invariant residues Blue = conserved residues

PAZ domainPAZ domain Conserved aromatic Conserved aromatic

residues that bind residues that bind the two-nucleotide the two-nucleotide 3’ overhang of an 3’ overhang of an siRNA are present in siRNA are present in PfAgo.PfAgo.

Side chains occupy Side chains occupy similar positions in similar positions in space, but they are space, but they are anchored to peptide anchored to peptide backbone in backbone in different locations.different locations.

Green = hAgo1 residues

So…where does the 3’ So…where does the 3’ overhang of the siRNA bind???overhang of the siRNA bind???

Right Here!!!

PAZ Domain ComparisonPAZ Domain Comparison

LL263263 and I and I261261 assume role of L assume role of L337337 and T and T335335 in in hAgo1, which anchor sugar ring of terminal hAgo1, which anchor sugar ring of terminal residue through vand der Waals interactions.residue through vand der Waals interactions.

WW213213 assume role of F assume role of F292 292 in hAgo1, which in hAgo1, which stacks against the terminal nucleotide.stacks against the terminal nucleotide.

RR220220 is positioned similarly to K is positioned similarly to K313313 that that contacts the penultimate nucleotide.contacts the penultimate nucleotide.

Reasoned that the PAZ domain in Reasoned that the PAZ domain in PfAgo binds RNA 3’ ends, as do PAZ PfAgo binds RNA 3’ ends, as do PAZ domains of fly and human domains of fly and human Argonautes.Argonautes.

PIWI is an RNase H DomainPIWI is an RNase H Domain

The domains are topologically identical

Five-stranded mixed β sheet surrounded by helices

PIWI DomainPIWI Domain

3 highly conserved catalytic 3 highly conserved catalytic carboxylatescarboxylates

One is located in One is located in ββ1, and one is 1, and one is located at C terminus of fourth strand located at C terminus of fourth strand ββ44

The third carboxylate variesThe third carboxylate varies Only requirement is a reasonable Only requirement is a reasonable

spatial position at the active site.spatial position at the active site.

Active Site Rotated 180°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 Active site is positioned in a cleft in the middle of the crescent in the the middle of the crescent in the groove below the PAZ domain.groove below the PAZ domain.

Here on overall

structure

Ago is SlicerAgo is Slicer Argonaute is the enzyme in RISC that Argonaute is the enzyme in RISC that

cleaves the mRNA.cleaves the mRNA. RNase H enzymes cleave ssRNA “guided” RNase H enzymes cleave ssRNA “guided”

by DNA strand in RNA/DNA hybrid.by DNA strand in RNA/DNA hybrid. Argonautes might do RNA cleavage Argonautes might do RNA cleavage

guided by the siRNA strand in a dsRNA guided by the siRNA strand in a dsRNA substrate.substrate.

Depends on MgDepends on Mg+2+2, like other RNase H , like other RNase H enzymes.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 bindingPossible substrate binding

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

siRNA guide siRNA guide interacts with PAZ interacts with PAZ cleftcleft

Model for siRNA-guided mRNA Model for siRNA-guided mRNA cleavagecleavage

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 From studies of other RNase H enzymes, expected that Argonaute enzymes, expected that Argonaute senses the minor groove width of the senses the minor groove width of the dsRNA, which differs from that of dsRNA, which differs from that of dsDNA.dsDNA.

Fits with RISC’s inability to cut DNA Fits with RISC’s inability to cut DNA substrates.substrates.

Opening of the claw might assist Opening of the claw might assist binding of mRNA – hinge region exists binding of mRNA – hinge region exists in interdomain connector at residues in interdomain connector at residues 317-320.317-320.

Added SupportAdded Support

In mammalian system, performed In mammalian system, performed mutational analysis on hAgo2mutational analysis on hAgo2

Conserved active site aspartates Conserved active site aspartates were altered = loss of nuclease were altered = loss of nuclease activity but retained siRNA binding.activity but retained siRNA binding.

Remaining Questions?Remaining Questions?

Other determinants beyond catalytic Other determinants beyond catalytic triad of PIWI domain that determine triad of PIWI domain that determine activity toward RNA substrates, such activity toward RNA substrates, such as conformational differences.as conformational differences.

Interactions with other factors may Interactions with other factors may be needed to create fully active be needed to create fully active SlicerSlicer

Identification of catalytic center of Identification of catalytic center of RISC awaited a drive towards RISC awaited a drive towards understanding RNAi at a structural understanding RNAi at a structural level.level.

A full understanding of the A full understanding of the underlying mechanism of RNAi will underlying mechanism of RNAi will need to be derived from a need to be derived from a combination of biochemical and combination of biochemical and structural studies of RISC.structural studies of RISC.