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Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

Domingues-Hamdi E, Vasseur C, Fournier JB, Marden MC, Wajcman H, Baudin-Creuza V - PLoS ONE (2014)

Bottom Line: SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression.The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition.The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.

ABSTRACT
Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

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H helix of human α-globin.(A) H helix sequence of the different truncated α-globins. The symbols indicate the amino-acids interacting with β-globin within the same αβ dimer (+) and those interacting with the heme molecule (•) [14]. The (x) symbol indicates the amino-acids interacting with AHSP. (B) Three-dimensional view of α-Hb. The representation was obtained from crystallographic data of oxyHb (2HHB) using the Pymol software. The truncated regions of the H helix investigated in this work are represented in blue (α-Hb1-138), purple (α-Hb1-134), red (α-Hb1-126), orange (α-Hb1-123) and grey (α-Hb1-117). The GH corner is shown in yellow.
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pone-0111395-g001: H helix of human α-globin.(A) H helix sequence of the different truncated α-globins. The symbols indicate the amino-acids interacting with β-globin within the same αβ dimer (+) and those interacting with the heme molecule (•) [14]. The (x) symbol indicates the amino-acids interacting with AHSP. (B) Three-dimensional view of α-Hb. The representation was obtained from crystallographic data of oxyHb (2HHB) using the Pymol software. The truncated regions of the H helix investigated in this work are represented in blue (α-Hb1-138), purple (α-Hb1-134), red (α-Hb1-126), orange (α-Hb1-123) and grey (α-Hb1-117). The GH corner is shown in yellow.

Mentions: In human α-thalassemias, the reduced availability of α-globin chains may be due to several molecular mechanisms, the most common being gene deletion [11]. However, in the past few years, the number of α chain Hb variants discovered with point mutations, short deletions, or insertions which resulted in α-thalassemia phenotypes, has increased [11]. Variants in which the contact region between α and β-subunits is altered, were often considered as the cause of these α-thalassemia phenotypes. The discovery of AHSP allows to consider an alternate explanation of some non-deletional α-thalassemias by impairing interaction between α-Hb and AHSP (Figure 1A). In a first study, it has been reported that two elongated α-Hb variants, Hb Constant Spring and Paksé associated with an α-thalassemia phenotype led to an impaired binding to AHSP [12]. Beside the elongated chains, point mutations with a single amino acid change were reported also to cause this clinical presentation. Our group has shown that Hb Groene Hart [α119(H2)Pro→Ser], a variant common in North Africa and in Southern Italy, causes a defect in the association with AHSP and behaves as an α+-thalassemia with two functional α-genes [13]. In a similar way, Lacerra et al described Hb Foggia [α117(GH5)Phe →Ser] an α-2 gene variant that led to an α-thalassemia phenotype in the carriers [14]. The mutated mRNA α-globin of this variant was present at normal level in the reticulocytes but no abnormal chains were detected suggesting that it was synthesized but very rapidly degraded likely due to impaired interaction with AHSP [14]. Recent studies, from our group and from others, demonstrated a similar defect in AHSP binding for an increasing number of α-Hb variants [15], [16]. The region 95–137 of α-Hb, composed by G and H helices, plays a predominant role in the interaction between α-Hb and AHSP [6]. In addition, the region 103–124 allows together the binding to AHSP and to β-Hb. Some amino-acids interact with AHSP, others only with β-Hb within an α1β1 dimer [17] and three (103, 117, 119) are involved in both contacts as shown in the figure 1A.


Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

Domingues-Hamdi E, Vasseur C, Fournier JB, Marden MC, Wajcman H, Baudin-Creuza V - PLoS ONE (2014)

H helix of human α-globin.(A) H helix sequence of the different truncated α-globins. The symbols indicate the amino-acids interacting with β-globin within the same αβ dimer (+) and those interacting with the heme molecule (•) [14]. The (x) symbol indicates the amino-acids interacting with AHSP. (B) Three-dimensional view of α-Hb. The representation was obtained from crystallographic data of oxyHb (2HHB) using the Pymol software. The truncated regions of the H helix investigated in this work are represented in blue (α-Hb1-138), purple (α-Hb1-134), red (α-Hb1-126), orange (α-Hb1-123) and grey (α-Hb1-117). The GH corner is shown in yellow.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219717&req=5

pone-0111395-g001: H helix of human α-globin.(A) H helix sequence of the different truncated α-globins. The symbols indicate the amino-acids interacting with β-globin within the same αβ dimer (+) and those interacting with the heme molecule (•) [14]. The (x) symbol indicates the amino-acids interacting with AHSP. (B) Three-dimensional view of α-Hb. The representation was obtained from crystallographic data of oxyHb (2HHB) using the Pymol software. The truncated regions of the H helix investigated in this work are represented in blue (α-Hb1-138), purple (α-Hb1-134), red (α-Hb1-126), orange (α-Hb1-123) and grey (α-Hb1-117). The GH corner is shown in yellow.
Mentions: In human α-thalassemias, the reduced availability of α-globin chains may be due to several molecular mechanisms, the most common being gene deletion [11]. However, in the past few years, the number of α chain Hb variants discovered with point mutations, short deletions, or insertions which resulted in α-thalassemia phenotypes, has increased [11]. Variants in which the contact region between α and β-subunits is altered, were often considered as the cause of these α-thalassemia phenotypes. The discovery of AHSP allows to consider an alternate explanation of some non-deletional α-thalassemias by impairing interaction between α-Hb and AHSP (Figure 1A). In a first study, it has been reported that two elongated α-Hb variants, Hb Constant Spring and Paksé associated with an α-thalassemia phenotype led to an impaired binding to AHSP [12]. Beside the elongated chains, point mutations with a single amino acid change were reported also to cause this clinical presentation. Our group has shown that Hb Groene Hart [α119(H2)Pro→Ser], a variant common in North Africa and in Southern Italy, causes a defect in the association with AHSP and behaves as an α+-thalassemia with two functional α-genes [13]. In a similar way, Lacerra et al described Hb Foggia [α117(GH5)Phe →Ser] an α-2 gene variant that led to an α-thalassemia phenotype in the carriers [14]. The mutated mRNA α-globin of this variant was present at normal level in the reticulocytes but no abnormal chains were detected suggesting that it was synthesized but very rapidly degraded likely due to impaired interaction with AHSP [14]. Recent studies, from our group and from others, demonstrated a similar defect in AHSP binding for an increasing number of α-Hb variants [15], [16]. The region 95–137 of α-Hb, composed by G and H helices, plays a predominant role in the interaction between α-Hb and AHSP [6]. In addition, the region 103–124 allows together the binding to AHSP and to β-Hb. Some amino-acids interact with AHSP, others only with β-Hb within an α1β1 dimer [17] and three (103, 117, 119) are involved in both contacts as shown in the figure 1A.

Bottom Line: SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression.The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition.The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (Inserm) U779, Université Paris XI, Paris, France.

ABSTRACT
Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.

Show MeSH