Iron
Regulatory Protein 1 in
Complex with Ferritin IRE-mRNA: the Moonlighting Form of c-Aconitase
Michael Harden '14 and Marta Hamilton '14
Contents:
I.
Introduction
The
enzyme aconitase has an
essential
role
in the
Krebs cycle, and is
therefore very
important
for energy processing.
The
enzyme
binds a
single citrate molecule at its centrally
located active site, acting as
a catalyst for the formation of
isocitrate. The enzyme’s
active site contains a bound iron-sulfur cluster, which
is necessary
for the reaction to proceed.
Mammals
have two types of
aconitase:
a
mitochondrial version that is
responsible for
most of the Krebs cycle functionality,
and
a
cytosolic
version (c-aconitase, shown here)
that synthesizes isocitrate for other
purposes.
However, the iron-sulfur cluster cannot
assemble
under iron
starved conditions, in which
case the cytosolic enzyme undergoes a
dramatic conformational change. This reorganized form of c-aconitase is
called iron regulatory protein 1 (IRP1), and has a completely different
functional role than c-aconitase.
IRP1
binds to iron-responsive elements (IREs), stem structures in
the untranslated regions of certain mRNAs. IRP1 binding to a
5’ IRE blocks ribosome binding, whereas binding to a
3’ IRE increases mRNA stability by preventing nuclease
degradation. Because aconitase only “moonlights” as
IRP1 in the absence of the iron-sulfer cluster, the presence of iron is
the signal that regulates the translation of these mRNAs.
Although
IRP1 interacts with a large number of mRNA transcripts, the
complex shown here is IRP1 bound with the IRE of Ferritin-encoding
mRNA.
II.
General Structure of IRP1:IRE -
The Transition from c-Aconitase
c-Aconitase
contains four domains with four distinct hydrophobic cores.
Upon
transitioning to IRP1, domains 1
and 2
become the central core of the
protein.
Although
domains 3
and 4
interact with each other in the
c-aconitase form, the two domains separate from each other and rotate
around the central core during rearrangement. This
rotation allows the
protein to assume the “L shape” of IRP1.
Transitioning
to this new
conformation opens up a large cavity, the
site of IRE-RNA binding. This opening of the protein reveals previously
hidden residues, many of which facilitate IRE-mRNA binding.
Domain
3 undergoes the most extensive conformational changes during this
rotation, a shift driven by a conformational change in the linker
region that connects domains 3 and 4. In c-aconitase, residues 593 to
614 in the linker region form two α-helices, separated by a
bend formed by proline residue 606. However, the transition to IRP1
causes this length of residues to form one long, continuous helix, a
change that enables domain 3’s dramatic repositioning.
III.
General Structure of Ferretin H IRE
The
ferritin IRE in this complex is comprised of 30 ribonucleotide residues
which form a helical stem-loop structure.
The
IRE contains 11
ordinary
Watson-Crick base pairs,
and
one
“wobble pair”
between U5
and G26.
The
loop at the top of the structure is made up of the three unpaired bases
A15,
G16,
and U17.
While U17 stacks with the molecule’s other
base pairs, the syn conformation of G16 allows it to stack with A15 as
they protrude out from the helix to face the protein. This conformation
causes a sharp bend in the RNA backbone.
This
important AGU loop
sequence is set apart
from the rest of the molecule by the
base pairing
of C14
and G18,
a pair of highly conserved bases in ferritin mRNAs.
Interestingly,
despite
this conservation, neither C14 nor G18 contacts
the protein, implying that their importance is mainly due to their
contribution to the structure of the IRE-mRNA.
One
other unpaired base, C8,
is important for RNA-protein interactions. C8
completely extends away from the RNA backbone, allowing it to make
extensive contacts with IRP1.
IV.
Interactions Between IRP1 and IRE-mRNA
There
are two major sites of contact between the IRE and IRP1: the site that
interacts with the AGU loop at the top of the mRNA, and the site that
interacts with the protruding C8 on the stem of the RNA.
The
These two
sites are distant enough from each other that their processes of
binding to IRP1 are completely unrelated.
As
mentioned previously, A15 and G16 extend away from the IRE-mRNA
and
make contact with IRP1 at the loop-binding pocket. Here, A15
and G16
form critical base-specific hydrogen bonds with Ser371
and Lys379
respectively.
The
complex is
strengthened by Van der Waals interactions
between each base and other residues in the cavity. U17
also contacts
the protein by forming a hydrogen bond with Arg269.
These
base-specific
interactions contribute to the sequence-selective binding of IRP1 to
IRE-containing mRNAs. Additional bonds between the RNA backbone and the
residues Asn535,
Thr438,
Asn439,
and Asn298
add to the strength
of the
IRP1-loop interaction.
C8
also makes contact with IRP1, and is the basis of the binding between
the protein and the stem of the IRE. C8 contacts the protein by being
inserted into a small cavity between residues Arg713
and Arg780.
From
this pocket, C8 forms hydrogen bonds with 4 separate residues: Ser681,
Pro682,
Asp781,
and Trp782.
Additionally,
the Arg780
residue is located
close enough to C8
to form an ionic bond with its phosphate group.
Of
these protein residues, only the serine residue bonds with
C8’s nitrogenous base, and the other three interact with the
RNA backbone. Nevertheless, this pocket of IRP1 greatly favors binding
to cytosine. This specificity is likely due to the tight fit of the
pocket: its very specific shape sterically prohibits other bases.
The
lower stem of the RNA contacts the protein at several other positions,
although they are less conserved among IREs than C8. One of the other
bases on the lower stem that interacts with the protein is G26, one of
the bases in the “wobble pair” mentioned
previously. The IRP1 residue Asn685
fits into the minor groove of the
RNA
to hydrogen bond with G26.
This
interaction is
possible because of the
partial displacement of G26 by its abnormal base pairing.
V.
IRP1's Selectivity for IRE-mRNA
As
mentioned previously, there are two separate focal points for the
binding of IRP1 to the IRE-mRNA: the AGU loop, and the protruding C8 on
the stem. The presence of two independent binding sites is a very
efficient way to guarantee that IRP1 does not accidentally interact
with other mRNA structures. This two-site mechanism might also allow
the protein to partially inhibit/induce translation of IRE-mRNAs that
are not a perfect fit for one of the binding sites. In this way, IRP1
might be able to use the same signal of iron starvation to exhibit
unique responses to different transcripts.
VI.
References
Artymiuk PJ, Green J. 2005.
The Double Life of Aconitase.
Structure 14(1):2-4.
Dupuy J, Volbeda A,
Carpentier P, Darnault C, Moulis J, Fontecilla-Camps JC. 2006. Crystal
Structure of Human Iron Regulatory Protein 1 as Cytosolic Aconitase.
Structure 14(1):129-39.
Hentze MW, Kuhn LC. 1996.
Molecular control of vertebrate iron metabolism: mRNA-based regulatory
circuits operated by iron, nitric oxide, and oxidative stress.
Proc Natl Acad Sci
93(16):8175-82.
*Walden WE, Selezneva AI,
Dupuy J, Volbeda A, Fontecilla-Camps JC, Theil EC, Volz K. 2006.
Structure of Dual Function Iron Regulatory Protein 1 Complexed with
Ferritin IRE-RNA. Science
314:1903-8.
*The primary source of
structural information for this tutorial
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