Characterization of Heme-Regulated eIF2R Kinase: Roles of the N-Terminal Domain in the Oligomeric State, Heme Binding, Catalysis, and Inhibition+
Abstract
Heme-regulated eIF2R 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 10-3 s-1) than for myoglobin (8.4 10-7 s-1) or the R-subunit of hemoglobin (7.1 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 domaintruncated 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 (eIF2R). 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 (IC50 ) 20 íM).