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. 2013 Jun 11;110(24):9722-7.
doi: 10.1073/pnas.1221743110. Epub 2013 May 28.

Pirin is an iron-dependent redox regulator of NF-κB

Fange Liu  1 , Imran Rehmani, Shingo Esaki, Rong Fu, Lirong Chen, Vesna de Serrano, Aimin Liu
Affiliations

Pirin is an iron-dependent redox regulator of NF-κB

Fange Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Pirin is a nuclear nonheme Fe protein of unknown function present in all human tissues. Here we describe that pirin may act as a redox sensor for the nuclear factor κB (NF-κB) transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to proinflammatory signals and controls the expression of a vast array of genes involved in immune and stress responses. Pirin's regulatory effect was tested with several metals and at different oxidations states, and our spectroscopic results show that only the ferric form of pirin substantially facilitates binding of NF-κB proteins to target κB genes, a finding that suggests that pirin performs a redox-sensing role in NF-κB regulation. The molecular mechanism of such a metal identity- and redox state-dependent regulation is revealed by our structural studies of pirin. The ferrous and ferric pirin proteins differ only by one electron, yet they have distinct conformations. The Fe center is shown to play an allosteric role on an R-shaped surface area that has two distinct conformations based on the identity and the formal redox state of the metal. We show that the R-shaped area composes the interface for pirin-NF-κB binding that is responsible for modulation of NF-κB's DNA-binding properties. The nonheme Fe protein pirin is proposed to serve as a reversible functional switch that enables NF-κB to respond to changes in the redox levels of the cell nucleus.

Keywords: coregulator; metalloprotein; oxidative stress; reactive oxygen species (ROS); signal transduction activation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The ferric, not ferrous, form of pirin substantially facilitates binding of p65 to the IgκB gene in SPR spectroscopy. (A) Detection of apo-, Fe(II)-, and Fe(III)-pirin effects on p65 binding to the IgκB site. (B) Response unit as a function of the pirin (1–290):p65 (19–291) ratio. (C) SPR spectroscopy with Fe(III)- and metal-substituted pirin proteins. (D) Effect of exogenous pirin on native NF-κB of the nuclear extract of HeLa cells. (E) QCM-D spectroscopy of p65 alone (red trace) and along with Fe(III)-pirin in 1:1–1:10 ratio (indicated by 1×, 3×, 5×, and 10×). (F) Comparison of the ΔD/Δf slope in a QCM-D study that indicates a conformational difference of the protein–DNA complex. The SPR traces are color coded with p65 only (red), p65 with Fe(II)-pirin (black), Fe(III)-pirin (blue), metal-striped pirin (purple), Co(II)-pirin (pink), Mn(II)-pirin (dark yellow), Mn(III)-pirin (wine), and Ni(II)-pirin (olive). Fe(III)-pirin alone (cyan) is shown in A and HeLa nuclear extract only (navy) is shown in D as controls, respectively.
Fig. 2.
Fig. 2.
The function of pirin on p65 is reversible depending on the redox state of the iron center. (A) Determination of the reduction potential of pirin by film cyclic voltammetry in an anaerobic chamber. (B) X-band EPR spectroscopy shows reversibility of the Fe(II)/Fe(III) couple in response to l-ascorbate. Shown are Fe(II)-pirin (250 µM, trace 1) after oxidation by O2 for 30 min (or 1 equivalent of H2O2 for 1 min, trace 2) and addition of 1 mM l-ascorbate for 5 min (trace 3) and for 60 min (trace 4). (C) l-ascorbate was introduced to the system, after forming the supercomplex from initial injection of p65/Fe(III)-pirin, at concentrations of 0 (black), 0.25 (navy), 2.5 (magenta), and 25 mM (green) at 980 s.
Fig. 3.
Fig. 3.
(A) Structural alignment of ferric (active toward p65) and ferrous (inactive) pirin. Colored regions indicate areas of deviation (≥1 Å), whereas gray regions are identical. The structural differences of Fe(III)-pirin are shown in yellow and those of Fe(II)-pirin are in green. (B) the zoom-in view highlights the deviation of a special R-shaped area with distinct conformations. (C) Superimposed Fe center structure of ferric (yellow), ferrous (green), and l-ascorbate–reduced (cyan) pirin structures.
Fig. 4.
Fig. 4.
(A–C) Structural differences shown in the alignment of ferric pirin (yellow) (A) with Co(II)-substituted pirin (pink), (B) with Mn(II)-pirin (red), and (C) with Mn(III)-pirin (blue). Colored regions indicate areas of deviation (≥1 Å), and gray regions indicate identical structural features.
Fig. 5.
Fig. 5.
(A) In silico docking model of pirin (green/yellow)–p65 (blue)–IgκB (golden/blue) supercomplex built from the corresponding crystal structures (Materials and Methods). The R-shaped region is highlighted in yellow and the Fe ion is represented in the sphere (ferric in yellow and ferrous in light green). (B) Zoomed-in view of the pirin (upper, green)–p65 (lower, cyan) interface region shows multiple complementary ion-pair interactions, including K34-E234, E32-R273, R14-E279, and R23-E282 (pirin-p65).
Fig. 6.
Fig. 6.
Mutation at the R-shaped surface impairs the ability of pirin to enhance the DNA-binding ability of p65 to the IgκB site. (A) SPR assay using p65 and immobilized IgκB with R23E (black trace), E32V (cyan), K34V (wine), and S13R/R14W (magenta). The nonsurface mutant Q115N (dark yellow) was used as a positive control. (B) Crystal structure of E32V aligned with native pirin structure. Colored regions indicate areas of deviation (≥1 Å), and gray regions indicate identical structural features.
Fig. 7.
Fig. 7.
Proposed function of human pirin. This model depicts a nonheme iron- and oxidation state-dependent regulation mechanism of NF-κB in the cell nucleus.
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