CRYSTAL STRUCTURE ANALYSIS OF MODULE-SUBSTITUTED CHIMERA-HEMOGLOBIN(F133V)

Tsuyoshi Shirai1, Masahiro Fujikake1, Takashi Yamane1, Kenji Inaba2, Koichiro Ishimori2, Isao Morishima2

1 Department of Biotechnology, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, JAPAN
2 Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501,

Keywords: protein evolution, heme protein, protein engineering, exon-shuffling

We constructed a chimera ba-subunit of hemoglobin in which a peptide segment (residues 97-146) that almost corresponds to module M4 of the b-subunit of human hemoglobin was substituted by the a-subunit counterpart (residues 92-141)1. The module M4 is encoded by a single exon in several hemoglobin genes. Amino acid residues of a-subunit that compose the a1b1 interface are localized mainly to M4 module. The chimera subunits typically attached to the b-subunits. Thus, the b-subunit binding function of the module M4 was successfully transplanted into the chimera ba-subunit using the "artificial exon-shuffling".

For an X-ray crystallographic study of the chimera subunit, a site-directed mutagenesis, aimed at removing a probable steric hindrance within the subunit, was introduced. A residue site of the chimera ba-subunit for fine-tuning mutagenesis was selected using a computer modeling method. A molecular model of the chimera ba-subunit was constructed based on the crystal structure of human hemoglobin A. The total energy of the model was minimized in order to reduce the hindrance of atoms and correct the arbitrary peptide bond at the b-a junction. As a result, Phe133 was found to be a main instabilizer of the structure, and the residue was replaced with Val in fine-tuned F133V subunit.

The chimera ba(F133V) subunit was crystallized into a carbonmonoxy form. X-ray diffraction data of the (CO-chimera ba[F133V])4 crystal were collected to a resolution of 2.5 A. The crystal belongs to the monoclinic space group P21 with the similar lattice constants (a = 62.9, b = 81.3, c = 55.1 A and b = 91.0o) with that of the crystal of hemoglobin H (b-subunit tetramer)3. Similarity of the packing in the crystals was confirmed by molecular replacement solutions of the crystals. The atomic model of (CO-chimera ba[F133V])4 was constructed based on the molecular replacement solution. The model shows that the implanted module M4 maintains the structure in the a-subunit, implying rigidity of the module. As expected, the chimera-subunit tetramer resembles hemoglobin H, although details of the subunit interface show a significant difference.

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