The N-terminal region of the heme-regulated eIF2alpha kinase is an autonomous heme binding domain.

The N-terminal domain (NTD) of the heme-regulated eukaryotic initiation factor (eIF)2alpha kinase (HRI) was aligned to sequences in the NCBI data base using ENTREZ and a PAM250 matrix. Significant similarity was found between amino acids 11-118 in the NTD of rabbit HRI and amino acids 16-120 in mammalian alpha-globins. Several conserved amino acid residues present in globins are conserved in the NTD of HRI. His83 of HRI was predicted to be equivalent to the proximal heme ligand (HisF8) that is conserved in all globins. Molecular modeling of the NTD indicated that its amino acid sequence was compatible with the globin fold. Recombinant NTD (residues 1-159) was expressed in Escherichia coli. Spectral analysis of affinity purified recombinant NTD indicated that the NTD contained stably bound hemin. Mutational analysis indicated that His83 played a critical structural role in the stable binding of heme to the NTD, and was required to stabilize full length HRI synthesized de novo in the rabbit reticulocyte lysate. These results indicate that the NTD of HRI is an autonomous heme-binding domain, with His83 possibly serving as the proximal heme binding ligand.     

Autophosphorylation of heme-regulated eukaryotic initiation factor 2α kinase and the role of the modification in catalysis

Heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), functions in response to heme shortage in reticulocytes and aids in the maintenance of a heme:globin ratio of 1:1. Under normal conditions, heme binds to HRI and blocks its function. However, during heme shortage, heme dissociates from the protein and autophosphorylation subsequently occurs. Autophosphorylation comprises a preliminary critical step before the execution of the intrinsic function of HRI; specifically, phosphorylation of Ser-51 of eIF2α to inhibit translation of the globin protein. The present study indicates that dephosphorylated mouse HRI exhibits strong intramolecular interactions (between the N-terminal and C-terminal domains) compared to phosphorylated HRI. It is therefore suggested that autophosphorylation reduces the intramolecular interaction, which induces irreversible catalytic flow to the intrinsic eIF2α kinase activity after heme dissociates from the protein. With the aid of MS, we identified 33 phosphorylated sites in mouse HRI overexpressed in Escherichia coli. Phosphorylated sites at Ser, Thr and Tyr were predominantly localized within the kinase insertion region (16 sites) and kinase domain (12 sites), whereas the N-terminal domain contained five sites. We further generated 30 enzymes with mutations at the phosphorylated residues and examined their catalytic activities. The activities of Y193F, T485A and T490A mutants were significantly lower than that of wild-type protein, whereas the other mutant proteins displayed essentially similar activity. Accordingly, we suggest that Tyr193, Thr485 and Thr490 are essential residues in the catalysis.     

Co-ordinate control of synthesis of mitochondrial and non-mitochondrial hemoproteins: a binding site for the HAP1 (CYP1) protein in the UAS region of the yeast catalase T gene (CTT1).

Control of expression of the Saccharomyces cerevisiae CTT1 (catalase T) gene by the HAP1 (CYP1) gene, a mediator of heme control of mitochondrial cytochromes, was studied. Expression of a CTT1-lacZ fusion in a hap1 mutant showed that the CTT1 promoter is under HAP1 control. As demonstrated by a gel retardation assay, the HAP1 protein binds to a heme control region of the CTT1 gene. This binding in vitro is stimulated by hemin. The HAP1-binding sequence was localized by using DNA fragments spanning different regions, by DNase I footprinting and by methylation interference of DNA-protein binding. The binding site was compared to the HAP1-binding sequences previously characterized in detail (UAS1CYC1, UASCYC7). There is strikingly little similarity between the three sequences, which have only four of those 23 bp in common which are protected from DNase I digestion. However, the pattern of major and minor groove contacts in the complex is quite similar in all three cases. The results obtained show that there is true co-ordinate control of expression of mitochondrial cytochromes and at least some extra-mitochondrial hemoproteins. Heme acts as a metabolic signal in this coordination, which is mediated by the HAP1 protein.     

Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins

Heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) 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, eIF2alpha, 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 Cys-Pro 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.     

Derivation of the globins from type b cytochromes

Summary Similarities in the amino acid sequences of vertebrate and invertebrate globins, b₅ and b₂ cytochromes and chicken sulfite oxidase point to a common ancestry for all of these proteins. The distal heme ligand (histidine or its equivalent) is common to both sets of proteins, but the proximal histidine ligand of the cytochromes is replaced by another histidine residue in the globins. This explains why the heme is reversed between globins and b₅ cytochromes. It seems likely that the genes for primitive globins contained three exons, the first two of which were derived from a cytochromelike DNA sequence. A model is presented to show how globins may have evolved from a pre-existing type bcytochrome; the complexity of the required changes is an indication that all globins are monophyletic.     

Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.     

Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand,hemin in its catalytic kinase domain

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.     

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition

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.     

Two heme-binding domains of heme-regulated eukaryotic initiation factor-2alpha kinase. N terminus and kinase insertion

In heme deficiency, protein synthesis in reticulocytes is inhibited by activation of heme-regulated alpha-subunit of eukaryotic initiation factor-2alpha (eIF-2alpha) kinase (HRI). Previous studies indicate that HRI contains two distinct heme-binding sites per HRI monomer. To study the role of the N terminus in the heme regulation of HRI, two N-terminally truncated mutants, Met2 and Met3 (deletion of the first 103 and 130 amino acids, respectively), were prepared. Met2 and Met3 underwent autophosphorylation and phosphorylated eIF-2alpha with a specific activity of approximately 50% of that of the wild type HRI. These mutants were significantly less sensitive to heme regulation both in vivo and in vitro. In addition, the heme contents of purified Met2 and Met3 HRI were less than 5% of that of the wild type HRI. These results indicated that the N terminus was important but was not the only domain involved in the heme-binding and heme regulation of HRI. Heme binding of the individual HRI domains showed that both N terminus and kinase insertion were able to bind hemin, whereas the C terminus and the catalytic domains were not. Thus, both the N terminus and the kinase insertion, which are unique to HRI, are involved in the heme binding and the heme regulation of HRI.     

Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2alpha kinase

The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.     

Activation of heme-regulated eukaryotic initiation factor 2alpha kinase by nitric oxide is induced by the formation of a five-coordinate NO-heme complex: optical absorption, electron spin resonance, and resonance raman spectral studies

Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.     

Multiple domains mediate heme control of the yeast activator HAP1

The activity of the yeast activator HAP1in vivo requires heme. A heme responsive domain of HAP1 was identified previously. It is adjacent to the DNA-binding domain, and appears to block DNA binding in the absence of heme. Here we describe a novel genetic selection which yielded mutants of HAP1 that are independent of heme when assayed for activation. These mutants define a second region of HAP1, close to the activation domain, which also controls its response to heme.     

Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene

We show that the yeast HAP1 activator locus encodes a protein that binds in vitro to the upstream activation site, UAS1, of the CYC1 gene (iso-1-cytochrome c). Binding of wild-type HAP1 and truncated HAP1 derivatives to UAS1 is evident in crudely fractionated yeast extracts using the gel electrophoresis DNA binding assay. The binding of HAP1 in vitro, like the activity of UAS1 in vivo, is stimulated by heme. HAP1 binds to region B, one of two portions of UAS1 shown to be important by genetic analysis of the site. Surprisingly, HAP1 binds to the same sequence as a second factor, RC2. Both HAP1 and RC2 bind to the same side of the helix, and make similar but not identical major and minor groove contacts that span two full turns. An additional factor that binds to the second important part of UAS1, the region A factor (RAF), is also identified. A model depicting the interplay of HAP1, RC2, and RAF in the control of UAS1 is presented.     

Phosphorylation of a heme-regulated eukaryotic initiation factor 2α kinase enhances the interaction with heat-shock protein 90 and substantially upregulates kinase activity

Heme-regulated eukaryotic initiation factor 2α kinase (HRI) functions under conditions of heme shortage caused by blood diseases such as erythropoietic protoporphyria and β-thalassemia, and retains the heme:globin ratio at 1:1 by sensing the heme concentration in reticulocytes. This HRI function is regulated by various factors including autophosphorylation and protein-protein interactions. A heat-shock protein controls HRI function, however, the molecular mechanism of catalytic regulation of HRI by the heat-shock protein is unclear. In the present study, we examined the interactions of HRI with a heat-shock protein, Hsp90, under various conditions, using a pull-down assay and measuring catalytic activity. It was found that [1] an interaction between Hsp90 and phosphorylated HRI was evident, whereas no interaction was observed between Hsp90 and HRI dephosphorylated by treatment with λ protein phosphatase; [2] Hsp90 enhanced the kinase activity of phosphorylated HRI but not dephosphorylated HRI, but this enhancement was not observed in the presence of heme; and, [3] autophosphorylation of HRI was not influenced by Hsp90. Therefore, we propose that autophosphorylation of HRI is critical for catalytic regulation by Hsp90 under heme-shortage conditions.