Abstract
Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn2+, revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRE-RNA, inhibiting mRNA activity; Fe2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn2+ (50 µM, 25 °C) increased IRE-RNA/IRP1 binding (Kd) 12-fold (FRT IRE-RNA) or 6-fold (ACO2 IRE-RNA); enthalpic contributions decreased ~61% (FRT) or ~32% (ACO2), and entropic contributions increased ~39% (FRT) or ~68% (ACO2). IRE-RNA/IRP1 binding changed activation energies: FRT IRE-RNA 47.0 ± 2.5 kJ/mol, ACO2 IRE-RNA 35.0 ± 2.0 kJ/mol. Mn2+ (50 µM) decreased the activation energy of RNA-IRP1 binding for both IRE-RNAs. The observations suggest decreased RNA hydrogen bonding and changed RNA conformation upon IRP1 binding and illustrate how small, conserved, sequence differences among IRE-mRNAs selectively influence thermodynamic and kinetic selectivity of the protein/RNA interactions.
Highlights
Cellular iron homeostasis is accomplished by the coordinated and balanced expression of iron storage protein ferritin and the iron-uptake protein transferrin receptor
Kd values for the binding of IRP1 to FRT iron responsive elements (IREs)-RNA increased from 4.6 ± 0.2 nM to 19.2 ± 0.4 nM, and for ACO2 IRE-RNA from 55.9 ± 3 nM to 155 ± 4 nM at temperatures ranging from 5 °C to 30 °C (Table 1)
The dissociation constant for the FRT IRE-RNA-Mn2+/IRP1 complex increased from 83.3 ± 4 to 193 ± 5 nM while the dissociation constant of ACO2 IRE-RNA-Mn2+/IRP1 complex increased from 535 ± 33 nM to 801 ± 36 at temperatures ranging from 5 °C to 30 °C (Table 1)
Summary
Cellular iron homeostasis is accomplished by the coordinated and balanced expression of iron storage protein ferritin and the iron-uptake protein transferrin receptor. Facile conformation change is important to IRP1 function, its interconversion between cytosolic aconitase and IRE-RNA binding protein. This interconversion involves large scale domain repositioning and localized conformational changes to form the two RNA binding pockets. We have previously shown[28, 29] that metal ions directly affect the IRE-RNA/IRP1 stability and the binding affinity favors ferritin IRE-RNA over aconitase IRE-RNA, illustrating the effects of phylogenetically conserved differences[30] between the two RNAs. Further, Fe2+ (used anaerobically to prevent formation of insoluble hydroxides) could bind to IRE-RNA and act as a ligand to reduce IRP1 binding[28]. Metal ion induced conformational change of IRP1/IRE-RNA complex is not known
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