Crystal Structures of HbA2 and HbE and Modeling of Hemoglobin δ4: Interpretation of the Thermal Stability and the Antisickling Effect of HbA2 and Identification of the Ferrocyanide Binding Site in Hb‡Click to copy article linkArticle link copied!
- Udayaditya Sen
- Jhimli Dasgupta
- Debi Choudhury
- Poppy Datta
- Abhijit Chakrabarti
- Sudipa Basu Chakrabarty
- Amit Chakrabarty
- Jiban K. Dattagupta
Abstract

Hemoglobin A2 (α2δ2) is an important hemoglobin variant which is a minor component (2−3%) in the circulating red blood cells, and its elevated concentration in β-thalassemia is a useful clinical diagnostic. In β-thalassemia major, where there is β-chain production failure, HbA2 acts as the predominant oxygen deliverer. HbA2 has two more important features. (1) It is more resistant to thermal denaturation than HbA, and (2) it inhibits the polymerization of deoxy sickle hemoglobin (HbS). Hemoglobin E (E26Kβ), formed as a result of the splice site mutation on exon 1 of the β-globin gene, is another important hemoglobin variant which is known to be unstable at high temperatures. Both heterozygous HbE (HbAE) and homozygous HbE (HbEE) are benign disorders, but when HbE combines with β-thalassemia, it causes E/β-thalassemia which has severe clinical consequences. In this paper, we present the crystal structures of HbA2 and HbE at 2.20 and 1.74 Å resolution, respectively, in their R2 states, which have been used here to provide the probable explanations of the thermal stability and instability of HbA2 and HbE. Using the coordinates of R2 state HbA2, we modeled the structure of T state HbA2 which allowed us to address the structural basis of the antisickling property of HbA2. Using the coordinates of the δ-chain of HbA2 (R2 state), we also modeled the structure of hemoglobin homotetramer δ4 that occurs in the case of rare HbH disease. From the differences in intersubunit contacts among β4, γ4, and δ4, we formed a hypothesis regarding the possible tetramerization pathway of δ4. The crystal structure of a ferrocyanide-bound HbA2 at 1.88 Å resolution is also presented here, which throws light on the location and the mode of binding of ferrocyanide anion with hemoglobin, predominantly using the residues involved in DPG binding. The pH dependence of ferrocyanide binding with hemoglobin has also been investigated.
‡
The coordinates of HbE, HbA2, and HbA2FC have been submitted to the RCSB as entries 1NQP, 1SI4, and 1SHR, respectively.
§
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics.
‖
Biophysics Division, Saha Institute of Nuclear Physics.
⊥
Thalassemia Hospital.
*
To whom correspondence should be addressed. Telephone: +91-033-2321-4986. Fax: +91-033-2337-4637. E-mail: jiban@ cmb2.saha.ernet.in.
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