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.
Uma
S
S
Department of Biochemistry, Oklahoma State University, Stillwater, USA.
Matts
R L
RL
Guo
Y
Y
White
S
S
Chen
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D006418:Heme; D006639:Histidine; D019892:eIF-2 Kinase; D001616:beta-Galactosidase