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 ␣-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, ␣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 solution experiments. We have constructed HbS Twin Peaks (␤Glu-63 Val, ␣Leu-1133 His) to ascertain the hitherto unknown role of the ␣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 found to be about 1.8-fold higher than HbS. To further authenticate the participation of the ␣113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the ␣113 mutation was coupled individually with two established fiber contact sites (␣16 and ␣23) located in the AB region of the ␣-chain: HbS(␣Lys-163 Gln, ␣Leu-1133 His), HbS(␣Glu-233 Gln, ␣Leu-1133 His). The single mutants at ␣16/␣23 sites were also engineered as controls. The C sat values of the HbS point mutants involving sites ␣16 or ␣23 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 ␣113 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.Sickle cell anemia is a consequence of a point mutation (Glu-63 Val) at the sixth position in the ␤-chain of the hemoglobin molecule (1). The replacement of a charged residue with a hydrophobic one on the surface of the protein drastically reduces the solubility of the deoxygenated sickle hemoglobin (HbS) 1 , leading to its polymerization into long helical fibers that are responsible for the clinical manifestations of sickle cell disease. Electron microscopy and crystallographic studies have suggested that both the deoxy HbS crystal and fiber are constructed from the same "Wishner-Love" double strands (2-5). The model of the fiber structure derived from these analyses consists of seven Wishner-Love double strands (6 -9).The polymerization process is triggered by lateral interactions of the donor Val-6␤ of a tetramer of one strand of the double strand with the acceptor pocket at the EF corner (elicited mainly by ␤Phe-85 and ␤Leu-88) of the ␤-chain of an adjacent molecule present in the second strand of the double strand. Subsequent intra-double strand and inter-double strand interactions involving several amino acid residues from both ␣-and ␤-chains contribute to the stabilization of the fiber structure. The polymerization-impairing o...

Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

“…Semi-synthesis of Mutant ␣-Chains-The complementary fragments of ␣-globin, ␣1-30 and ␣31-141, were obtained by subjecting the V8 protease digest of ␣-globin to Sephadex G50 chromatography (24). The sequence comprising ␣1-30 with Gly or Aib residues at positions 19 and 21 was assembled by solid-phase peptide synthesis.…”

“…The respective purified peptides were ligated with the complementary fragment (␣31-141) derived from human or langur chains by V8 protease-mediated ligation reaction (23,24). Semi-synthetic mutant globins were purified by CM52 chromatography in the presence of 8 M urea as described previously (24). ESMS, tryptic peptide mapping, and amino acid sequencing were employed to establish the chemical identity of the mutant chains.…”

“…The sequence comprising ␣1-30 with Gly or Aib residues at positions 19 and 21 was assembled by solid-phase peptide synthesis. The respective purified peptides were ligated with the complementary fragment (␣31-141) derived from human or langur chains by V8 protease-mediated ligation reaction (23,24). Semi-synthetic mutant globins were purified by CM52 chromatography in the presence of 8 M urea as described previously (24).…”

“…We have earlier successfully employed a semi-synthetic reaction, namely, V8 protease-mediated coupling of ␣1-30 and ␣31-141 fragments to produce ␣141 (␣-globin), for the construction of mutant ␣-subunits (23,24). This strategy, although restrictive in nature, is especially suited for the incorporation of non-coded Aib residues and, in appropriate situations, could be effectively utilized for the introduction of 20 standard amino acids.…”

“…Deoxygenated sickle hemoglobin (HbS) polymerizes into long helical fibers that are believed to be responsible for the pathophysiology of the sickle cell disease. The knowledge gleaned so far from structural analysis of HbS crystal (2)(3)(4), solution polymerization studies of natural variants or engineered mutant hemoglobins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), and electron microscopic studies (25) have led to a 14-stranded model of the fiber (26,27). These strands appear as seven double strands of the type found in HbS crystals, albeit with a slight twist caused by fiber packing.…”