Regulatory Protein 1 in
Complex with Ferritin IRE-mRNA: the Moonlighting Form of c-Aconitase
Michael Harden '14 and Marta Hamilton '14
enzyme aconitase has an
Krebs cycle, and is
for energy processing.
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
for the reaction to proceed.
have two types of
mitochondrial version that is
most of the Krebs cycle functionality,
version (c-aconitase, shown here)
that synthesizes isocitrate for other
However, the iron-sulfur cluster cannot
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.
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.
IRP1 interacts with a large number of mRNA transcripts, the
complex shown here is IRP1 bound with the IRE of Ferritin-encoding
General Structure of IRP1:IRE -
The Transition from c-Aconitase
contains four domains with four distinct hydrophobic cores.
transitioning to IRP1, domains 1
become the central core of the
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.
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.
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.
General Structure of Ferretin H IRE
ferritin IRE in this complex is comprised of 30 ribonucleotide residues
which form a helical stem-loop structure.
IRE contains 11
Watson-Crick base pairs,
loop at the top of the structure is made up of the three unpaired bases
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.
important AGU loop
sequence is set apart
from the rest of the molecule by the
a pair of highly conserved bases in ferritin mRNAs.
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.
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.
Interactions Between IRP1 and IRE-mRNA
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.
sites are distant enough from each other that their processes of
binding to IRP1 are completely unrelated.
mentioned previously, A15 and G16 extend away from the IRE-mRNA
make contact with IRP1 at the loop-binding pocket. Here, A15
form critical base-specific hydrogen bonds with Ser371
strengthened by Van der Waals interactions
between each base and other residues in the cavity. U17
the protein by forming a hydrogen bond with Arg269.
interactions contribute to the sequence-selective binding of IRP1 to
IRE-containing mRNAs. Additional bonds between the RNA backbone and the
add to the strength
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
this pocket, C8 forms hydrogen bonds with 4 separate residues: Ser681,
residue is located
close enough to C8
to form an ionic bond with its phosphate group.
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.
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
to hydrogen bond with G26.
possible because of the
partial displacement of G26 by its abnormal base pairing.
IRP1's Selectivity for IRE-mRNA
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.
Artymiuk PJ, Green J. 2005.
The Double Life of Aconitase.
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.
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
*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
*The primary source of
structural information for this tutorial
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