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Biological characteristics of two lysines on human serum albumin in the high‐affinity binding of 4 Z ,15 Z ‐bilirubin‐IXα revealed by phage display
Author(s) -
Minomo Ai,
Ishima Yu,
KraghHansen Ulrich,
Chuang Victor T. G.,
Uchida Makiyo,
Taguchi Kazuaki,
Watanabe Hiroshi,
Maruyama Toru,
Morioka Hiroshi,
Otagiri Masaki
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08316.x
Subject(s) - human serum albumin , stereochemistry , chemistry , mutant , ligand (biochemistry) , bovine serum albumin , pichia pastoris , recombinant dna , biochemistry , receptor , gene
4 Z ,15 Z ‐bilirubin‐IXα (4 Z ,15 Z ‐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 4 Z ,15 Z ‐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 4 Z ,15 Z ‐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 4 Z ,15 Z ‐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 4 Z ,15 Z ‐BR was transformed into highly water‐soluble isomers at a much faster rate than the M‐form. In addition, the M‐form of 4 Z ,15 Z ‐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 4 Z ,15 Z ‐BR, Lys199 plays a more prominent role in the elimination of 4 Z ,15 Z ‐BR. DatabaseModel data are available in the PMDB database under accession numbers 2VUE and 2BXA .