Elucidation of the Heme Binding Site of Heme-regulated Eukaryotic Initiation Factor 2α Kinase and the Role of the Regulatory Motif in Heme Sensing by Spectroscopic and Catalytic Studies of Mutant Proteins

Abstract

Heme-regulated eukaryotic initiation factor 2␣ (eIF2␣) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2␣, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory CysPro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.Eukaryotic cells decrease their overall rates of protein synthesis for survival in response to a variety of stress conditions, such as shortage of amino acids, UV light illumination, virus infection, and accumulation of denatured proteins. Much of the decrease in protein synthesis is caused by phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) 4 at Ser-51 by eIF2␣ kinases that respond specifically to stress (1-4). Heme-regulated eIF2␣ kinase or heme-regulated inhibitor (HRI) is a member of the eIF2␣ kinase family that controls globin synthesis in response to the heme concentration in reticulocytes (5-8). HRI is inactive at normal heme concentrations. Under conditions of heme deficiency, the enzyme is activated by autophosphorylation and subsequently phosphorylates eIF2␣ at Ser-51. In addition to globin, HRI controls the synthesis of tryptophan 2,3-dioxygenase and cytochrome P450 2B in liver upon acute porphyria (9, 10). Thus, HRI is possibly one of the most important existing heme sensor protein families for eukaryote survival in response to cell emergency states.Heme-responsive/sensing proteins, also known as "heme sensor proteins" are a current focus of investigation. In these proteins heme association (or dissociation) per se regulates various important physiological functions, such as transcription, proteolysis, and kinase activity (11...

Identification and Functional and Spectral Characterization of a Globin-coupled Histidine Kinase from Anaeromyxobacter sp. Fw109-5

“…Samples were loaded on a 7.5% SDS poly-acrylamide gel containing 50 M Phos-tag acrylamide and 0.1 mM MnCl 2 . Phosphorylated protein interacted with the Phos-tag manganese complex, so that mobility was slower than that of phosphatefree protein (38,39). Proteins were visualized with Coomassie Brilliant Blue R350 (GE Healthcare) staining.…”

“…We examined the autophosphorylation activities of various heme complexes of AfGcHK using Phos-tag SDS-PAGE, which differentiates between non-phosphorylated and phosphorylated proteins (38,39). The Fe(III), Fe(II)-O 2 , and Fe(II)-CO complexes clearly displayed autophosphorylation activity but not the Fe(II) complex (Fig.…”

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

“…It is clear, however, that when HRI becomes activated in vivo, its function creates unfavorable replicative conditions for the baculovirus that can be reversed by viral expression of PK2 protein. Based on the restricted phylogenetic distribution of key N-terminal histidine residues and cysteine/proline motifs implicated in the recognition of heme by HRI (26,27), we speculate that the ancestral function of HRI-like genes was antiviral. This model is consistent with evolutionary analysis suggesting that the heme responsiveness of HRI appeared only after the split of ancestral insects and metazoans.…”

Heme Sensor Proteins

“…The heme-regulated inhibitor (HRI; eIF2␣ kinase 1) phosphorylates eIF2␣ during heme deprivation or oxidative stress (39). In heme-replete cells, HRI binds heme with Cys/His coordination and is inactive (40). At low heme levels, the heme dissociates, and HRI autophosphorylates and then phosphorylates eIF2 (41).…”