Two Heme-binding Domains of Heme-regulated Eukaryotic Initiation Factor-2α Kinase

Abstract

In heme deficiency, protein synthesis in reticulocytes is inhibited by activation of heme-regulated ␣-subunit of eukaryotic initiation factor-2␣ (eIF-2␣) 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-2␣ 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.

Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein

“…This interpretation is not limited to the situation with isolated peptides, since recombinant IRP2 also failed to bind haem immobilized on cross-linked agarose ( Figure 6) and to produce a low-spin EPR signal with the cofactor. It is noteworthy that not all sequences containing a potential HRM have the ability to bind haem, as shown for the C-terminal portion of the haem-regulated α-subunit of eukaryotic initiation factor-2 kinase [40].…”

Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases

“…HRI is a multidomain protein composed of five structural domains (Figure 2), namely, the N-terminal domain (NTD), the kinase I domain (K-I), the kinase insertion domain (KI), the kinase II domain (K-II) and the C-terminal domain (CTD) [56,[59][60][61]. The catalytic domain of HRI is divided in two halves, one on each side of the KI domain, and activation of HRI is thought to involve bringing residues on each of these two halves of the catalytic domain into close proximity [23,56,[59][60][61]. Two HRMs are present in HRI, one in the KI domain and the other in the CTD [23,56,[59][60][61].…”

“…The catalytic domain of HRI is divided in two halves, one on each side of the KI domain, and activation of HRI is thought to involve bringing residues on each of these two halves of the catalytic domain into close proximity [23,56,[59][60][61]. Two HRMs are present in HRI, one in the KI domain and the other in the CTD [23,56,[59][60][61]. The heme binding site in the KI domain is thought to reversibly bind heme and to play a role in the coordination of HRI activity with changes in heme concentration [61].…”

“…Two HRMs are present in HRI, one in the KI domain and the other in the CTD [23,56,[59][60][61]. The heme binding site in the KI domain is thought to reversibly bind heme and to play a role in the coordination of HRI activity with changes in heme concentration [61]. The second heme binding site, contained in the NTD without an HRM, consists of a stably bound heme that co-purifies with HRI [59,62].…”

“…The isolated NTD stably binds heme but does not display any kinase activity. The CTD domain does not bind to heme, although it contains an HRM motif [61].…”