Characterization of Heme-Regulated eIF2α Kinase:  Roles of the N-Terminal Domain in the Oligomeric State, Heme Binding, Catalysis, and Inhibition

Abstract

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.

Eukaryotic initiation factor 2α kinase is a nitric oxide‐responsive mercury sensor enzyme: Potent inhibition of catalysis by the mercury cation and reversal by nitric oxide

“…In particular, the decrease in absorption at 366 nm is characteristic of Hg 2+ competition with heme. Absorption at around 370 nm was suggested to involve in the thiol-Fe 3+ -hemin bond of heme-sensor proteins [11,15]. We propose that the metal ions trigger a switch from the axial Cys ligand bound to heme to a water molecule or OH-anion, resulting in a decrease in absorption at 366 nm.…”

“…A plasmid encoding (His) 6 -tagged wild-type HRI comprising residues 1-619 (His 6 HRI) from mouse liver cDNA was originally constructed using the pET28 vector (Novagen, Madison, WI, USA), as described previously [6,11]. We introduced a PreScissione protease (Amersham Biosciences, Piscataway, NJ, USA) recognition site in the expression vector to remove the (His) 6 -tag.…”

“…(His) 6 -tagged HRI was expressed in Escherichia coli BL21 (DE3) Codon Plus RIL (Stratagene, La Jolla, CA, USA) harboring pET28a-PreScission/HRI, and purified using a previous protocol [6,11]. The (His) 6 tag was cut with PreScissione protease, according to the manufacturer's protocol.…”

“…Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Phosphorylated proteins were detected by immunoblotting with an anti-phosphorylated eIF2a antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) [11].…”

“…recovery Since the N-terminal domain of HRI is important for sensing the heme concentration [11], we determined whether it plays a role in the reversal of Hg 2+ -mediated inhibition by NO using a truncated protein lacking the first 145 amino acids. Hg 2+ inhibited the activity of truncated and full-length HRI to a similar extent.…”