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 (K_d) 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.

The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNA∙IRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 10⁶ M⁻¹ and 1.0, respectively. Addition of Fe²⁺(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNA∙IRP1. Further, thermodynamic parameters of APP mRNA∙IRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (–25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNA∙IRP1 also confirmed the complex formation, having the rate of association (k_on) and the rate of dissociation (k_off) as 341 μM⁻¹ s⁻¹, and 11 s⁻¹, respectively. The addition of Fe²⁺ has decreased the rate of association (k_on) by ~ three-fold, whereas the rate of dissociation (k_off) has increased by ~ two-fold. The activation energy for APP mRNA∙IRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe²⁺ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNA∙IRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNA∙IRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions.

Interaction of iron responsive elements (IRE) mRNA with the translational machinery is an early step critical in the initiation of protein synthesis. To investigate the binding specificity of IRE mRNA for eIF4F, kinetic rates for the eIF4F·IRE RNA interactions were determined and correlated with the translational efficiency. The observed rate of eIF4F·FRT IRE RNA interactions was 2-fold greater as compared to eIF4F·ACO2 IRE RNA binding. Addition of iron enhanced the association rates and lowered the dissociation rates for the eIF4F binding to both IRE RNAs, with having higher preferential binding to the FRT IRE RNA. The binding rates of both eIF4F·IRE RNA complexes correlated with the enhancement of protein synthesis in vitro. Presence of iron and eIF4F in the depleted WGE significantly enhanced translation for both IRE RNAs. This suggests that iron promotes translation by enhancing the binding rates of the eIF4F∙IRE RNA complex. eIF4F·IRE RNA binding is temperature-dependent; raising the temperature from 5 to 25°C, enhanced the binding rates of eIF4F·FRT IRE (4-fold) and eIF4F·ACO2 IRE (5-fold). Presence of Fe²⁺ caused reduction in the activation energy for the binding of FRT IRE and ACO2 IRE to eIF4F, suggesting a more stable platform for initiating protein synthesis. In the presence of iron, lowered energy barrier has leads to the faster association rate and slower rate of dissociation for the protein-RNA complex, thus favoring efficient protein synthesis. Our results correlate well with the observed translational efficiency of IRE RNA, thereby suggesting that the presence of iron leads to a rapid, favorable, and stable complex formation that directs regulatory system to respond efficiently to cellular iron levels.

α-Synuclein (α-Syn) is implicated in the pathophysiology of Parkinson’s disease (PD) and plays a significant role in neuronal degeneration. Iron response proteins (IRPs) bind to iron response elements (IREs) found in the 5′-untranslated regions (5′-UTRs) of the messenger RNA that encode the α-Syn gene. This study used multi-spectroscopic approach techniques to investigate the impact of iron on α-Syn IRE RNA binding to IRP1. The formation of a stable complex between α-Syn RNA and IRP1 was suggested by fluorescence quenching results. Fluorescence measurements showed that α-Syn RNA and IRP1 had a strong interaction, with a binding constant (K_a) of 21.0 × 10⁶ M⁻¹ and 1:1 binding stoichiometry. About one binding site per IRP1 molecule was suggested by the α-Syn RNA binding. The K_a for α-Syn RNA•IRP1 with added Fe²⁺ (50 μM) was 6.4 μM⁻¹. When Fe²⁺ was added, the K_a of α-Syn RNA•IRP1 was reduced by 3.3 times. These acquired K_a values were used to further understand the thermodynamic characteristics of α-Syn RNA•IRP1 interactions. The thermodynamic properties clearly suggested that α-Syn RNA binding to IRP1 was an entropy-favored and enthalpy-driven event, with significant negative ΔH and small positive ΔS. For α-Syn RNA•IRP1, the Gibbs free energy (ΔG) was −43.7 ± 2.7 kJ/mol, but in the presence of Fe²⁺, it was −36.3 ± 2.1 kJ/mol. These thermodynamic calculations indicated that hydrogen bonding as well as van der Waals interactions might help to stabilize the complex formation. Additionally, far-UV CD spectra verified α-Syn RNA•IRP1 complex formation, and α-Syn RNA and Fe²⁺ induce secondary structural alteration of IRP1. According to our findings, iron alters the hydrogen bonding in α-Syn RNA•IRP1 complexes and induces a structural change in IRP1. This suggests that iron selectively affects the thermodynamics of these RNA–protein interactions.

The hallmark of Alzheimer’s disease (AD) is the buildup of amyloid-β (Aβ), which is produced when the amyloid precursor protein (APP) misfolds and deposits as neurotoxic plaques in the brain. A functional iron responsive element (IRE) RNA stem loop is encoded by the APP 5′-UTR and may be a target for regulating the production of Alzheimer’s amyloid precursor protein. Since modifying Aβ protein expression can give anti-amyloid efficacy and protective brain iron balance, targeted regulation of amyloid protein synthesis through modulation of 5′-UTR sequence function is a novel method for the prospective therapy of Alzheimer’s disease. Numerous mRNA interference strategies target the 2D RNA structure, even though messenger RNAs like tRNAs and rRNAs can fold into complex, three-dimensional structures, adding even another level of complexity. The IRE family is among the few known 3D mRNA regulatory elements. This review seeks to describe the structural and functional aspects of IREs in transcripts, including that of the amyloid precursor protein, that are relevant to neurodegenerative diseases, including AD. The mRNAs encoding the proteins involved in iron metabolism are controlled by this family of similar base sequences. Like ferritin IRE RNA in their 5′-UTR, iron controls the production of APP in their 5′-UTR. Iron misregulation by iron regulatory proteins (IRPs) can also be investigated and contrasted using measurements of the expression levels of tau production, Aβ, and APP. The development of AD is aided by iron binding to Aβ, which promotes Aβ aggregation. The development of small chemical therapeutics to control IRE-modulated expression of APP is increasingly thought to target messenger RNAs. Thus, IRE-modulated APP expression in AD has important therapeutic implications by targeting mRNA structures.