Homodimeric
Structure and Double-stranded RNA Cleavage Activity of the RNase
IIIb Domain of Human Dicer
Mary Myers '12 and Brian Thorne '12
Contents:
I.
Introduction
Dicer protein is responsible for generating short, non-coding
interfering
RNAs and
microRNAs duplexes from
dsRNA, and is therefore critical for the
RNAi pathway[1],
which leads to sequence-specific translational repression of genes.
The dsRNA
duplexes formed via Dicer cleavage are generated with symmetric
2–3nt 3' overhangs and 5'-phosphate and 3'-hydroxyl groups,
which trigger the degradation of mRNAs with a matching
sequence.[2]
Human Dicer is a large, multi
domain protein
about
200,000
Daltons in size, and is classified as a class 3 RNase
protein. Classification is based on
the observation of two RNase III nuclease
domains; RNase
IIIa and RNase
IIIb. Both
of these domains can be found either as fragments, or
can coordinate into homodimers.
While
this tutorial focuses predominately on
the RNase IIIb
domain of the protein, the folllowing image represents the
remaining domains
included in a complete dicer complex:
[3]
II.
General Structure
Analysis
has indicated that the nature of the single processing center in Human
(Hs)-Dicer, suggests that RNase IIIb domain fragments are capable of
forming intramolecular
dimers, which result in two catalytic sites within the processing
center; one on each end of the dimer interface.
Each catalytic
site
cleaves
RNA
phosphodiester bonds and generates ~20nt dsRNA
products with 2 nt 3' overhangs.
Other domains, such as the RNA Binding
domain (RBD), dsRNA Binding
Domain (dsRBD) and PAZ assist the cleavage reaction.
The RNase IIIb domains specifically requires Mg2+
ions to
catalyze the hydrolysis of the RNA phosphodiester backbone.
The
crystal
structure of RNase IIIb domain is
known to a resolution of 2.0
Å. Each of the monomers that constitute the RNase
IIIb homodimer possess a highly conserved structure with
62% helical
composition[3]
and
contains 8 α-helices as
well
as a relatively rare 310
helix (helix 2)[5],
but
has no
ß-strands in its structure. The
dimerization of the
monomers and the protein's specific folding result in a negatively
charged valley which supports
the Mg2+ ions required for
cleavage. This negative protein valley
is represented in the
accompanying image by the blue region
within the interface of the
dimer.
The charge difference within the valley provided by the
positive
metal ions allows the dsRNA substate to bind more readily by possibly
neutralizing the repulsive charge, and in addition results
in the resolution of two
RNA cutting sites within
each catalytic
site. Two cutting
sites are necessary at each catalytic
site, so that both strands of the dsRNA substrate can be processed, and
the 3' overhang can be formed.
III.
Homodimerization
The RNase IIIb exists in solution as a stable dimer with
tight associations. The interface
between the two monomers forms the
base of the previously mentioned
valley
via interactions between the anti-parallel
α3
helicies
the
and
the
α4
helicies,
as well as interactions between the α7
C-terminus
and
α8
N-terminus
within
each
monomer.
Hydrophobic interactions are the main driving force
of dimerization
through
the interactions of aromatic residues from both monomers in the dimer
interface formed by the two
anti-parallel α3
helicies :
[Phe]1706
interacts with [Thr]1717,
[Leu]1732,
and [Arg]1736.
-[Tyr]1714
interacts with [Ile]1711
and [Leu]1827:
The Leucine residues
increase dimer stability by associating the α9
helicies with
the dimer interface.
[Tyr]1721
interacts with [Arg]1703
and [Leu]1703
near the ends of the α3
helicies.
IV.
Active Site
As
mentioned, two Mg2+ ions
are bound by each
RNase
IIIb domain either at, or close to, the active site.
The
two ions are differentiated as Mg-1
and Mg-2
depending
on their
location within the active site. These four
ions are each
coordinated by eight interactions
with
oxygen atoms.
Mg-1 atoms directly
interact with carboxylate
oxygen atoms
in [Asp]1810,
[Glu]1705,
,
,
and [Glu]1813,
as
well
as three molecules of water,
.
[Asp]1709
interacts with one of the three coordinated water
molecules that
stabilize the Mg-1
ion. Mg-2
interacts directly with the carboxylate
oxygen of [Asp]1709
and four water
molecules
.
. [Asp]1709
also interacts with one
of the water molecules here as well
.
Two [Asp]1713
residues (only one shown) positioned
centrally in the homodimer interact
with the coordinated water
molecules
.
It
is presumed that the Mg-1
ion
functions to activate the
nucleophilic
water molecule, and is positioned appropriately within the
molecule for
it to serve this purpose.
Being
near the end of the catalytic
site places the nucleophile in the
most stereochemically favorable area
within this specific domain. When complexed in a crystal with
the
enzyme, the dsRNA substrate ended up
colliding with the N-terminus
of
the α6
helix
within the RNase IIIb domain.
Given
this information, a
comformational change is likely to
occur either in the protein or with
the dsRNA substrate; maybe even both. When the substrate is
within the dimer interface, the Mg-2 ions are closely associated with
the minor groove of the RNA helix between the two cleavage sites.
A logical explanation for this observation would be to
neutralize
the negatively charged valley within the dimer near the active sites.[3]
VI.
References
[1]http://www.scq.ubc.ca/wp-content/siRNA.gif
[2] Cioca, DP et al. 2003.
RNA interference is the functional pathway with therapeutic
potential in huma myeloid leukemia cell lines. Gene
Cancer Therapy. Feb;
10(2): 125-33.
[3]Takeshita, D. et al. 2007.
Homodimeric
Structure and
Double-stranded RNA Cleavage Activity of the C-terminal RNase III
Domain of Human Dicer. The
Journal of Molecular Biology
374:106-120.
[4]http://www.genecards.org/cgi-bin/carddisp.pl?gene=dicer1
[5]
Blaszcyk, J. et al. 2004. Noncatalytic Assembly of Ribonuclease III
with Double-Stranded RNA. Structure
12:3 March
2004,
Pages 457-466.
Back to Top