Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IX[alpha] revealed by phage display

• Published in: The FEBS journal 2011-11, Vol.278 (21), p.4100
• Main Authors: Minomo, Ai, Ishima, Yu, Kragh-Hansen, Ulrich, Chuang, Victor T. G, Uchida, Makiyo, Taguchi, Kazuaki, Watanabe, Hiroshi, Maruyama, Toru, Morioka, Hiroshi, Otagiri, Masaki
• Format: Article
• Language: English
• Subjects: Amino acids; Binding sites; Molecular biology; Proteins
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08316.x

4Z,15Z-bilirubin-IXα (4Z,15Z-BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domainII was constructed, after three rounds of panning against immobilized 4Z,15Z-BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positions195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domainII were expressed in Pichiapastoris for ligand-binding studies. The binding of 4Z,15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domainII proteins. The P-helicity conformer (P-form) of 4Z,15Z-BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M-form bound to the K199A mutants. Photoconversion experiments showed that the P-form of 4Z,15Z-BR was transformed into highly water-soluble isomers at a much faster rate than the M-form. In addition, the M-form of 4Z,15Z-BR showed higher affinity for domainI than for domainII. The present findings suggest that, whereas both Lys195 and Lys199 in subdomainIIA are important for the high-affinity binding of 4Z,15Z-BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z-BR. Database Model data are available in the PMDB database under accession numbers and . We identified key amino acid residues of HSA involved in 4Z,15Z-bilirubin-IXα (4Z,15Z-BR) binding using phage display analysis and point mutation techniques. From various binding assays and photoconversion experiments, whilst both Lys195 and Lys199 in subdomain IIA are important for the high affinity binding of 4Z,15Z-BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z-BR. [PUBLICATION ABSTRACT]