Location and bond type of intermolecular contacts in the polymerisation of haemoglobin S

Kwong, Suzanna; Benesch, Ruth E.; Edalji, Rohinton

doi:10.1038/269772a0

Cited by 72 publications

(37 citation statements)

References 23 publications

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“…On this basis many workers have concluded that Hb F does not copolymerize, in contrast to the results presented here, which suggest that substitution by a non-S hemoglobin can give rise to a polymer that has a different structure and an increased solubility. This hypothesis is supported by our previous finding that a tetramer composed of 3S chains and mutant a chains (a47 Asp-His) forms gels that have a much greater solubility and a lower hemoglobin concentration than those of Hb S (21). Moreover, recent electron microscopy studies in collaboration with R. Crepeau and S. Edelstein have shown that this double mutant hemoglobin forms fibers with very specific structural alterations, in which certain pairs of strands are missing and others are added (unpublished).…”

supporting

confidence: 69%

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Solubilization of hemoglobin S by other hemoglobins.

Edalji¹,

Benesch²,

Kwong³

1980

Proc. Natl. Acad. Sci. U.S.A.

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The polymerization of mixtures of Hb S with hemoglobins A, A2, and F has been investigated by analysis of the proportions of S and non-S hemoglobin both in the supernate and-in the pellet after centrifugation. In all cases the non-S hemoglobin was incorporated into the polymer even in the absence of hybrids in the order A > A2 > F. The solubility of Hb S is substantially increased by the other hemoglobins, especially by Hb F. which would account for its antisickling effect. It appears that the excluded volume effect of the other hemoglobin on Hb S is largely counterbalanced by the solubilizing effect arising from the interaction between the two hemoglobins in solution. The ability of hybrid hemoglobins to gel was demonstrated directly with tetramers in which afts dimers were covalently linked to aflA, a5A2, and ayF dimers.The so-called "sickle-sparing" effect of other hemoglobins on hemoglobin S (Hb S) was described more than 20 years ago by Singer and Singer (1) and by Allison (2), who found that some hemoglobins such as Hb A and Hb C greatly decrease the concentration of Hb S necessary for gelling, whereas Hb F had a much smaller effect. It was assumed by these authors and in much of the subsequent work with other hemoglobins (3-6) that the "sickle-sparing" hemoglobins act by substituting for Hb S in the polymer, i.e., by copolymerizing with it. By contrast, it was concluded that Hb F and, more recently, Hb A2 (7-9) do not copolymerize with Hb S.However, it has now been realized that another factor plays a major role in the gelling of Hb S in the presence of other hemoglobins (10-12). In these very concentrated and nonideal solutions the presence of any other hemoglobin [and, indeed, any The purpose of the work reported here was therefore to investigate the participation of Hb F and Hb A2 in the polymerization of Hb S by direct analysis, not only of the supernate, but also of the gel. In addition, hemoglobins in which an af3s dimer was covalently crosslinked to either an acy or an a6 dimer were used to test the ability of these mixed tetramers to polymerize. MATERIALS AND METHODSThe hemoglobins were isolated from hemolysates, prepared as described (19) from normal blood, sickle trait blood, and cord blood. After dialysis against 0.1 M glycine at pH 7.6, the hemolysates were fractionated on DEAE-cellulose, Whatman DE52 (20), under the conditions shown in Table 1. Sectional columns (Kontes Glass) were used and the desired hemoglobin was removed from the appropriate segment without elution of the column (21).Crosslinked (XL) hemoglobins were prepared by using the reagent 2-nor-2-formylpyridoxal 5'-phosphate as described (22,23). For the preparation of crosslinked mixed tetramers, the reaction was performed on equimolar amounts of Hb S and the other hemoglobin that had been mixed under oxygen to allow for hybridization. After the coupling reaction, which takes place under anaerobic conditions, the hemoglobins were converted to the cyanmet form by oxidation with 1.2 equivalents of potassium ferricyanide...

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supporting

confidence: 69%

“…After dialysis against 0.1 M glycine at pH 7.6, the hemolysates were fractionated on DEAE-cellulose, Whatman DE52 (20), under the conditions shown in Table 1. Sectional columns (Kontes Glass) were used and the desired hemoglobin was removed from the appropriate segment without elution of the column (21).…”

Section: Methodsmentioning

confidence: 99%

Solubilization of hemoglobin S by other hemoglobins.

Edalji¹,

Benesch²,

Kwong³

1980

Proc. Natl. Acad. Sci. U.S.A.

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“…The ␣113 site is of interest because of its unique structural location. First, ␣113 is in sequence contiguity with a cluster of GH corner residues, ␣114, ␣115, and ␣116, which are established or implicated intra-double strand contact sites of the HbS fiber (7,11,16). Second, the three-dimensional structure of the hemoglobin brings the AB region of the ␣-chain in close proximity to the GH corner.…”

mentioning

confidence: 99%

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

Sivaram

Sudha

Roy

2001

Journal of Biological Chemistry

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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...

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“…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

Journal of Biological Chemistry

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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...

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Location and bond type of intermolecular contacts in the polymerisation of haemoglobin S

Cited by 72 publications

References 23 publications

Solubilization of hemoglobin S by other hemoglobins.

Solubilization of hemoglobin S by other hemoglobins.

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

Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

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