A role for the alpha 113 (GH1) amino acid residue in the polymerization ofsickle hemoglobin - Evaluation of its inhibitory strength and interaction linkage with two fiber contact sites (alpha 16/23) located in the AB regionof the alpha-chain
Mvs. Sivaram et al., A role for the alpha 113 (GH1) amino acid residue in the polymerization ofsickle hemoglobin - Evaluation of its inhibitory strength and interaction linkage with two fiber contact sites (alpha 16/23) located in the AB regionof the alpha-chain, J BIOL CHEM, 276(21), 2001, pp. 18209-18215
A cluster of amino acid residues located in the AB-GH region of the alpha -
chain are shown in intra-double strand axial interactions of the hemoglobin
S (HbS) polymer. However, alpha Leu-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 sol
ution experiments. We have constructed HbS Twin Peaks (beta Glu-6-->Val, al
pha Leu-113-->His) to ascertain the hitherto unknown role of the alpha 113
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 fou
nd to be about 1.8-fold higher than HbS, To further authenticate the partic
ipation of the alpha 113 site in the polymerization process as well as to e
valuate its relative inhibitory strength, we constructed HbS tetramers in w
hich the alpha 113 mutation was coupled individually with two established f
iber contact sites (alpha 16 and alpha 23) located in the AB region of the
alpha -chain: HbS(alpha Lys-16-->Gln, alpha Leu-113-->His), HbS(alpha Glu-2
3-->Gln, alpha Leu-113-->His). The single mutants at alpha 16/alpha 23 site
s were also engineered as controls. The C-sat values of the HbS point mutan
ts involving sites alpha 16 or alpha 23 were higher than HbS but markedly l
ower as compared with HbS Twin Peaks. In contrast, C-sat values of both dou
ble mutants were comparable with or higher than that of HbS Twin Peaks, The
demonstration of the inhibitory effect of alpha 113 mutation alone or in c
ombination with other sites, in quantitative terms, unequivocally establish
es a role for this site in HbS gelation, These results have implications fo
r development of a more accurate model of the fiber that could serve as a b
lueprint for therapeutic intervention.