title: Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IXα revealed by phage display.
abstract: 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 domain II 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 positions 195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4Z,15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II 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 domain I than for domain II. The present findings suggest that, whereas 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.
authors: Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
authors: Ai
authors: A
authors: Minomo
authors: Yu
authors: Y
authors: Ishima
authors: Ulrich
authors: U
authors: Kragh-Hansen
authors: Victor T G
authors: VT
authors: Chuang
authors: Makiyo
authors: M
authors: Uchida
authors: Kazuaki
authors: K
authors: Taguchi
authors: Hiroshi
authors: H
authors: Watanabe
authors: Toru
authors: T
authors: Maruyama
authors: Hiroshi
authors: H
authors: Morioka
authors: Masaki
authors: M
authors: Otagiri
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001663:Bilirubin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D012709:Serum Albumin; D008239:Lysine; D001663:Bilirubin