Predicting the Structural Impact of the Hemoglobin Constant Spring Mutation on HBA2

Background: Thalassemia is a disease caused by the impaired production of globin chains. The most prevalent non-deletional α-thalassemia variants in Indonesia include Hemoglobin Constant Spring (Hb CS), Hb Adana, and codon 22 (GGC→GGT). However, structural bioinformatics studies on Hb CS remain limited. This study aims to predict the three-dimensional structure of Hb CS and its comparison to normal HBA2 globin chain structures.Materials and methods: The reference HBA2 nucleotide sequence was retrieved from NCBI and translated to amino acid using ExPASy Translate. The three-dimensional structure of Hb CS was predicted using Iterative Threading Assembly Refinement (I-TASSER). In addition, physicochemical properties, as well as structural and functional predictions, were analyzed using the ProtParam and PredictProtein servers.Results: I-TASSER generated five structural models of Hb CS, all showing high similarity to HBA2 (81.98% sequence identity; RMSD 0.322–0.396 Å). Compared with HBA2, Hb CS exhibited increased molecular weight, higher theoretical pI, and a higher instability index. The additional 31-amino-acid C-terminal segment was predicted to form a solvent-exposed intrinsically disordered region predominantly composed of coil structures. The additional amino acid segment was predicted to form a solvent-exposed intrinsically disordered region, suggesting reduced structural stability. Some gene ontology terms of Hb CS were found to be consistent with those of functional hemoglobin.Conclusion: Hb CS mutation causes an extension of hydrophobic amino acids and forms the IDP region, thereby reducing its stability in the cytoplasm compared to HBA2. This mutation also triggers the REMD mechanism that accelerates mRNA degradation due to excessive translation into the 3'-UTR region. Keywords: α-thalassemia, HBA2, Hb CS, in silico