Human Argonaute2
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Human Argonaute 2

Scott Watters '14 and Stephen Raithel '13


I. Introduction and General Structure


Gene silencing is one of a number of mechanisms for post transcriptional regulation of RNA within eukaryotic cells. Unique long double stranded RNA’s are processed into microRNA (miRNA) which are then uptaken by the RNA induced silencing complex (RISC). Specifically, miRNA is directly bound by a ribonucleoprotein particle (RNP) of the Argonaute (Ago) protein family (Meister et al, 2004).  Once the complementary RNA is bound,  Argonaute proteins either slice the RNA directly, or recruit additional factors to silence the RNA. 

All members of the Argonaute family have conserved PIWI and PAZ (PIWI-argonaute-zwille) domains. The PIWI domain has a structure very similar to that of the RNase H from bacteria suggesting that this domain is responsible for the endonucleolytic activity of RISC (Shirle & MacRae, 2012). PAZ has a number of hydrophobic and positively charged residues which account for both Ago affinity for RNA and it’s binding to the siRNA and miRNA processing protein Dicer (Filipowicz, 2005).

In humans, there are 4 different Ago proteins. Of these, only Argonaute 2 has been shown to have mRNA silencing activity (Filipowicz 2005). This protein has two domains in addition to PIWI  PAZ; these are  N and MID .  Ago2 also contains an N-terminal domain as well as two linker domains which connect PAZ to the N and MID domains; respectively, L1 L2 .

II. RNA Binding

Human Argonaute2 appears to hold seven nucleotides of the guide RNA in a fixed conformation ; this binding is stabilized by hydrogen bonds, contacts to the phosphate backbone of the RNA, and Van der Waals contacts with the sugar bases.

Many of the stabilizing contacts are through the MID and the PIWI domains. To be specific, Lysine 566 and Arginine 792 make an ion-dipole interaction with the phosphate on the backbone of the guide RNA, while Tyrosine 790 makes an H-bond to a phosphate .   Tyrosine 804, Serine 798, Lysine 709, and Histidine 753 additionally stabilize the phosphates in the RNA backbone.

Additional more minor contacts, include protein RNA interaction stabilization of this conformation with the 5’ RNA base stacking with Tyrosine 529 . Additionally, the 5’ phosphate of this base forms H-bonds with Tyrosine 529, Lysine 533, Glutamine 545, and Lysine 566 . All of these amino acids are located in the MID domain. 

None of these contacts are base specific, as you would expect for the protein to be able to bind siRNA of many different sequences.

III. RNA Specificity and Catalytic Activity

RNA target vs. DNA target

The 2’ hydroxyl of nucleotide 5 bonds to the amide on the backbone of Isoleucine 756 and the 2’ hydroxyl of nucleotide 7 bonds to the backbone carbonyl of Alanine 221 .  Additionally, the backbone carbonyls of Asparagine 562 and Arginine 792 make water mediated contacts to the 2’ hydroxyl of nucleotide 2 (bonds not shown due to lack of consistent water position data in crystal structure).  Overall, this amounts to very little additionally stabilization provided by the 2’ hydroxyl of the nucleotides.  In fact, DNA bases and 2’ fluoro substitutions do not prevent the binding of siRNAs to Argonaute2.




When bound to Argonaute2, the RNA nucleotides have the Watson-Crick faces (the edges of the nitrogenous bases farthest from the glycoside linkage) exposed to the exterior environment in an A-form conformation.  However, Isoleucine 365 is inserted between bases 6 and 7 which introduces a kink in the near A-form structure ; the minor-grove edge of nucleotide 7 is further stabilized in this kinked position by Methionine 364 .  Both Methionine 364 and Isoleucine 365 are located on alpha helix 7 on the L2 region of Argonaute2. 

Base pairing of nucleotide 7 to another nucleotide possibly shifts helix 7 , thus releasing the kink and allowing nucleotides 6 and 7 to base pair.  It is hypothesized that these are the reasons why effective pairing to nucleotide 7 is so crucial for miRNA targeting, and it is also thought that the protein could introduce a kink after the RNA has been sliced to allow it to dissociate from the protein.

RNase H Activity


The PIWI domain of argonaute2 contains the endonucleolytic active site of the molecule. As with the ribonucleases that PIWI shares homology with, three carboxylate residues are responsible for this catalytic activity; the R groups of D641, D669, and either E683 or E673 (Rivas et al, 2005). These residues coordinate with one of two Mg2+ ions which catalyze the hydrolysis of the 3’ phosphodiester bond of RNA (bonds to metal ions not shown as protein was not crystalized with Mg2+).

IV. Protein-Protein Interactions

The PIWI domain contains many of the residues responsible for argonaute’s protein-protein binding. A number of aliphatic amino acids engage in hydrophobic interactions with GW proteins (so called for their increased quantity of glycine and tryptophan residues). Two separate hydrophobic pockets interacts with tryptophan residues of an associate protein. In the first L650, I651, Y654, K660, L694, and Y698 pack against an inserted tryptophan . In the second, the side chains of F587, P590, V591, A620, F653, and F659 stack against a tryptophan , while the main chain carbonyl of F587 hydrogen bonds to the indole ring of tryptophan .

These two pockets are separated by a span of ~24 Å . This region has some elasticity and is roughly the same distance as the amino acid linker between the two inserting tryptophan residues of many GW proteins. It is thought that argonaute recognizes GW proteins through identification of tandem tryptophan, separated by this distance (Schirle & MacRae, 2012).

V. References

Filipowicz, W. (2005). RNAi: The Nuts and Bolts of the RISC Machine. Cell, 122(1), 17–20. doi:

Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., & Tuschl, T. (2004). Human Argonaute2 Mediates RNA Cleavage Targeted by miRNAs and siRNAs. Molecular Cell, 15(2), 185–197. doi:

Schirle, N. T., & MacRae, I. J. (2012). The Crystal Structure of Human Argonaute2. Science , 336 (6084 ), 1037–1040. doi:10.1126/science.1221551

Rivas, F. V, Tolia, N. H., Song, J.-J., Aragon, J. P., Liu, J., Hannon, G. J., & Joshua-Tor, L. (2005). Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol, 12(4), 340–349. Retrieved from

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