Human
Argonaute 2
Scott Watters '14 and Stephen
Raithel '13
Contents:
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.
BINDING
OTHER RNA
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:http://dx.doi.org/10.1016/j.cell.2005.06.023
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:http://dx.doi.org/10.1016/j.molcel.2004.07.007
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
http://dx.doi.org/10.1038/nsmb918
Back to Top