Role of Tyr residues in the contact region of anti-lysozyme monoclonal antibody HyHEL10 for antigen binding.
It has been shown that Tyr residues are unusually localized in the regions of antibodies responsible for contact with antigens (Padlan, E. A. (1990) Proteins Struct. Funct. Genet. 7, 112-124). In order to clarify the role of these Tyr residues in antigen binding, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, whose structure has been well studied in complex with its antigen, was investigated. Four Tyr residues in the VH chain (HTyr-33, HTyr-50, HTyr-53, and HTyr-58) were replaced with Ala, Leu, Phe, or Trp, and the interactions between these mutant Fv fragments and HEL were studied by inhibition assay of the enzymatic activity of HEL and isothermal titration calorimetry. Twelve mutant Fv fragments could be expressed, but two mutants (HY50W and HY58W) could not be obtained in the Escherichia coli expression system, and a further two mutants (HY33A and HY50A) could not be purified by affinity chromatography. It was shown by inhibition assay that Tyr residues at each mutated site made positive contributions to the interaction to different degrees. Thermodynamic studies showed that the role of Tyr residues in antigen binding was to obtain enthalpic energy. The roles of Tyr residues in antibody HyHEL10 for the association with antigen, HEL, can be summarized as follows: 1) formation of hydrogen bonds by the hydroxyl group, 2), creating more favorable interactions through the aromatic ring and decreasing the entropic loss upon binding, and 3) allowing hydrophobic interaction through the side chain. The four Tyr residues studied here were found to play significant roles in the association in various ways.
Tsumoto
K
K
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
Ogasahara
K
K
Ueda
Y
Y
Watanabe
K
K
Yutani
K
K
Kumagai
I
I
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology*; D001483:Base Sequence; D002151:Calorimetry; D002645:Chickens; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000276:immunology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D014443:Tyrosine; D009113:Muramidase