β-Chain contact sites in the haemoglobin S polymer

Nagel, Ronald L.; Johnson, Joyce E.; Bookchin, Robert M.; Garel, Marie-Claude; Rosa, J.; Schilirò, Gino; Wajcman, Henri; Labie, Dominique; Moo-Penn, Winston F.; Castro, Oswaldo

doi:10.1038/283832a0

Cited by 96 publications

(48 citation statements)

References 13 publications

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“…On the other hand, only deoxy-HbS generates polymers. As demonstrated by binary mixtures of HbS with Hb mutants (27), the deoxy-HbS fiber has as assembly unit, the Wishner-Love double strand (5,28). This structure requires strong up and down and lateral contacts between tetramers and also between double strands.…”

Section: Resultsmentioning

confidence: 99%

Liquid–liquid separation in solutions of normal and sickle cell hemoglobin

Galkin

Chen

Nagel

et al. 2002

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

191

176

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We show that in solutions of human hemoglobin (Hb)-oxy-and deoxy-Hb A or S-of near-physiological pH, ionic strength, and Hb concentration, liquid-liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40°C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid-liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.T he primary pathogenic event in sickle cell anemia (1) is the polymerization of the mutated hemoglobin (Hb) S into linear fibers (2), mostly in the postcapillary venules (3-5), forming spherulitic domains, sheaves of parallel polymers (6-8), and other secondary structures, which stretch the erythrocytes and increase the intracellular viscosity leading to vasoclussion (9). In the absence of a curative treatment (2, 10), the main impetus for research has been on slowing and preventing the polymerization of deoxy-HbS (11). Recent experiments (12), simulations (13), and theory (14) suggest that the kinetics of formation of protein solid phases can be controlled by shifting the phase boundary for liquid-liquid (L-L) separation, occurring with some proteins (15)(16)(17). In this article, we discuss the tests of the first prerequisite for the action of this control mechanism-the existence of a dense liquid phase in the solutions of normal and sickle cell Hb at near-physiological conditions. Methods Procedures for Direct Monitoring of L-L Separation and Nucleation ofPolymers. Samples of Hb solutions (A or S) with concentration in the range 9.6-35 g͞dl, in oxygenated or deoxygenated state, in 0.15 M potassium phosphate buffer at pH 7.35, with 0.1-1% (wt͞vol) polyethylene glycol (PEG) of molecular mass 8,000 g͞mol (PEG 8000) were held between two microscope slides. The solution layer thickness, determined by focusing on imperfections on the bottom and top inside glass surfaces, was 10-40 m and the sample volume was a few microliters. The slides were sealed with Maunt-Quick (Daido Sangyo, Japan) sealant, and mounted on a custom-made temperature control stage. The latter consists of an aluminum block attached to thermoelectric (Peltier, Sterling, MA) coolers and has an opening that allows the approach of the microscope condenser from the slide bottom for differential interference contrast (DIC) imaging. The controller ensures temperature stability and control within Ϯ0.05°C between Ϫ5 and 70°C. For the detection of the HbS polymers, polymer bundle domains, and gels, we used a DIC-equipped microscope (Leitz Orthoplan, magnification ϫ1,000) as in refs. 6, 7, and 18. To avoid heat loss through the microscope lens or the condenser, they were both inserted in brass coolers through which we flowed water coming from a temperature-contr...

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Section: Resultsmentioning

confidence: 99%

Liquid–liquid separation in solutions of normal and sickle cell hemoglobin

Galkin

Chen

Nagel

et al. 2002

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

191

176

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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.…”

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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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“…The mouse ␣-chain exhibits a very high inhibitory potential against ␤ S -dependent polymerization compared with any other single point mutations of the contact sites of deoxyHbS (3)(4)(5).…”

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confidence: 99%

“…Inasmuch as solution studies have identified Wishner-Love double strand as the assembly unit of the HbS polymer (3), it is legitimate to use the crystal structure of deoxyHbS to locate intra-double strand contact sites. Sites ␣16, ␣20, ␣23, and ␣116 comply with this characteristic and, in addition, are in spatial proximity despite the fact they are widely separated in the primary sequence (10).…”

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confidence: 99%

Inhibition of Sickle β-Chain (βS)-dependent Polymerization by Nonhuman α-Chains

Nacharaju

Roy

White

et al. 1997

Journal of Biological Chemistry

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Horse ␣-chain inhibits sickle ␤-chain-dependent polymerization; however, its inhibitory potential is not as high as that of mouse ␣-chain. Horse ␣-(1-30) and ␣-(31-141) segments make, respectively, minor and major contributions to the inhibitory potential of horse ␣-chain. The sum of the inhibitory potential of the two segments does not account for the inhibitory potential of the fulllength horse ␣-chain.Although the polymerization inhibitory potential of horse ␣-chain is lower than mouse ␣-chain, the inhibitory potential of horse ␣-(31-141) is comparable to that of mouse ␣-(31-141). When mouse ␣-(1-30) is stitched to horse ␣-(31-141), the product is a chimeric ␣-chain with an inhibitory potential greater than mouse ␣-chain. In contrast, the stitching of horse ␣-(1-30) with mouse ␣-(31-141) had no additional inhibitory potential.Molecular modeling studies of HbS containing the mouse-horse chimeric ␣-chain indicate altered sidechain interactions at the ␣ 1 ␤ 1 interface when compared with HbS. In addition, the AB/GH corner perturbations facilitate a different stereochemistry for the interaction of the ⑀-amino group of Lys-16(␣) with the ␤-carboxyl group of Asp-116(␣), resulting in a decrease in the accessibility of the side chain of Lys-16(␣) to the solvent. Based on molecular modeling, we speculate that these perturbations by themselves, or in synergy with the altered conformational aspects of the ␣ 1 ␤ 1 interactions, represent the molecular basis of the superinhibitory potential of the mouse-horse chimeric ␣-chains.

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β-Chain contact sites in the haemoglobin S polymer

Cited by 96 publications

References 13 publications

Liquid–liquid separation in solutions of normal and sickle cell hemoglobin

Liquid–liquid separation in solutions of normal and sickle cell hemoglobin

Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

Inhibition of Sickle β-Chain (βS)-dependent Polymerization by Nonhuman α-Chains

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