Integrative Biochemical and In Silico Characterization of Congenital Bisalbuminemia and Its Impact on Albumin Structure and Ligand Transport

Congenital bisalbuminemia is a rare inherited anomaly of human serum albumin (HSA) characterized by the coexistence of two electrophoretically distinct albumin fractions. Although generally asymptomatic, these variants offer a unique opportunity to investigate how single amino acid substitutions influence albumin’s structure, electrostatic properties, and ligand-binding behavior Two unrelated Tunisian pediatric cases exhibiting double albumin bands on capillary electrophoresis were investigated. Serum biochemical analysis, Sanger sequencing of the ALB gene, and comprehensive in silico modeling were performed. Molecular dynamics and docking simulations assessed the effects of the identified mutations—Christchurch (p.Arg24Gln) and Tradate-2 (p.Lys249Gln)—on protein folding, surface charge distribution, local flexibility, and binding affinities for key physiological ligands including fatty acids, thyroid hormones, and common drugs Both cases revealed heterozygous ALB mutations corresponding to previously described Christchurch (slow) and Tradate-2 (fast) variants. Structural modeling indicated that the Christchurch variant introduces an unprocessed signal peptide carrying an additional positive charge, while the Tradate-2 substitution decreases positive charge near the first binding cavity (C1), increasing net negativity. These changes subtly modify electrostatic potential and rigidity without altering global folding. Docking analyses using oleic, linoleic, palmitic, and stearic acids; triiodothyronine (T3) and thyroxine (T4); and ibuprofen, diclofenac, and diazepam revealed preserved but slightly altered ligand-binding affinities and minor shifts in binding-site localization within subdomains IIA and IIIA. These results demonstrate subtle redistribution of binding strength and polarity consistent with electrophoretic mobility differences observed experimentally This study reports, for the first time in Africa, two congenital bisalbuminemia variants characterized at biochemical, genetic, and in silico levels. It also represents the first structural assessment of their potential impact on albumin’s transport functions. The findings underscore how computational biochemistry complements clinical investigation in elucidating rare protein variants and highlight the translational relevance of albumin modeling for understanding drug transport, diagnostic assay interference, and potential therapeutic bioengineering.