The N-terminal region of the heme-regulated eIF2 alpha kinase is an autonomous heme binding domain

The N-terminal domain (NTD) of the heme-regulated eukaryotic initiation fac tor (eIF)2 alpha kinase (HRI) was aligned to sequences in the NCBI data bas e using ENTREZ and a PAM250 matrix. Significant similarity was found betwee n amino acids 11-118 in the NTD of rabbit HRI and amino acids 16-120 in mam malian alpha-globins. Several conserved amino acid residues present in glob ins are conserved in the NTD of HRI. His83 of HRI was predicted to be equiv alent to the proximal heme ligand (HisF8) that is conserved in all globins. Molecular modeling of the NTD indicated that its amino acid sequence was c ompatible with the globin fold. Recombinant NTD (residues 1-159) was expres sed in Escherichia coli. Spectral analysis of affinity purified recombinant NTD indicated that the NTD contained stably bound hemin. Mutational analys is indicated that His83 played a critical structural role in the stable bin ding of heme to the NTD, and was required to stabilize full length HRI synt hesized de novo in the rabbit reticulocyte lysate. These results indicate t hat the NTD of HRI is an autonomous heme-binding domain, with His83 possibl y serving as the proximal heme binding ligand.