A Recombinant Sickle Hemoglobin Triple Mutant with Independent Inhibitory Effects on Polymerization

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...

Section: Fig 5 Kinetics Of Polymerization Of Hbs Twinmentioning

confidence: 89%

A Role for the α113 (GH1) Amino Acid Residue in the Polymerization of Sickle Hemoglobin

Sivaram

Sudha

Roy

2001

“…In the current scenario, the definition of a fiber contact by both theoretical as well as experimental approach is centered on specific amino acid residues. The solution studies consider a given residue as a fiber contact only if the mutation of that site results in an altered polymerization behavior relative to the native HbS (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). On the other hand, the fiber models define a contact residue based on the distance; residues with contact distances of about 5 Å or less are generally considered as interacting partners (26,27).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

Sudha

Anantharaman

Sivaram

et al. 2004

The AB and GH regions of the ␣-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala 19 and Ala 21 of the AB corner with more flexible Gly or rigid ␣-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of ␣-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (␣Asn 78 3 His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of ␣Gln 78 , His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (␣Gln 78 3 His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His 78 . Similar synergistic coupling, between ␣His 78and an intra-double-strand contact point (␣16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with ␣78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly.Sickle cell anemia arises because of a point mutation at the sixth position of the ␤-chain (␤Glu 6 3 Val) in the hemoglobin (Hb) 1 molecule (1). 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 (2-4), solution polymerization studies of natural variants or engineered mutant hemoglobins (5-24), 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. The models specify several amino acid residues from both ␣-and ␤-chains that participate in inter-or intradouble-strand contacts stabilizing the fiber structure. By and large, the fiber models agree with the solution polymerization experiments of mutant hemoglobins in that polymerizationsensitive mutations are usually located in fiber contact regions (27). However, the models present an approximate...

“…Measurement of HbS Polymerization-The C sat /dextran assay of Bookchin, which was performed under completely anaerobic conditions, was used (27), and control values for HbS are 34 Ϯ 1 mg/ml. 1A where the ratio of the 11-residue peptide to HbS was 0.5).…”

Section: Methodsmentioning

confidence: 99%

“…For all of the experiments reported by Danish et al (28), peptide:HbS ratios did not exceed 4:1. Because the rationale for using a cross-linked peptide was not convincing to us, and the control uncross-linked 8-residue peptide had no effect on HbS polymerization, we decided to study the effect of our 11-residue peptide on HbS polymerization in the complete absence of O 2 (27). As shown in Fig.…”

Section: Effects Of Human Red Cell Membrane Band 3 Cytoplasmic Fragmementioning

confidence: 99%

Human Erythrocyte Membrane Band 3 Protein Influences Hemoglobin Cooperativity

Zhang

Manning

Falcone

et al. 2003

Self Cite

Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O 2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139 -237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O 2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.