A role for the α113 (GH1) amino acid residue in the polymerization of sickle hemoglobin

Abstract

A cluster of amino acid residues located in the AB-GH region of the α-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, αLeu-113 (GH1) located in the periphery is not implicated in any interactions by either crystal structure or models of the fiber, and its role in HbS polymerization has not been explored by solution experiments. We have constructed HbS Twin Peaks (βGlu-6→Val, αLeu-113→His) to ascertain the hitherto unknown role of the α113 site in the polymerization process. The structural and functional behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks polymerized with a slower rate compared with HbS, and its polymer solubility (C _sat) was found to be about 1.8-fold higher than HbS. To further authenticate the participation of the α113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the α113 mutation was coupled individually with two established fiber contact sites (α16 and α23) located in the AB region of the α-chain: HbS(αLys-16→Gln, αLeu-113→His), HbS(αGlu-23→Gln, αLeu-113→His). The single mutants at α16/α23 sites were also engineered as controls. The C _sat values of the HbS point mutants involving sites α16 or α23 were higher than HbS but markedly lower as compared with HbS Twin Peaks. In contrast,C _sat values of both double mutants were comparable with or higher than that of HbS Twin Peaks. The demonstration of the inhibitory effect of α113 mutation alone or in combination with other sites, in quantitative terms, unequivocally establishes a role for this site in HbS gelation. These results have implications for development of a more accurate model of the fiber that could serve as a blueprint for therapeutic intervention.