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Iron Regulatory Protein 1 in Complex with Ferritin IRE-mRNA: the Moonlighting Form of c-Aconitase

Michael Harden '14 and Marta Hamilton '14


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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