title: Molecular modeling and site-directed mutagenesis of an anti-phosphotyrosine antibody predicts the combining site and allows the detection of higher affinity interactions.
abstract: Bacterially expressed Fv regions of the anti-phosphotyrosine antibody Py20 have been shown to have an affinity similar to the parent IgG. Previous studies revealed a requirement for both heavy and light chain variable regions, VH and VK joining amino acids and the heavy chain D region for high affinity binding. In order to identify amino acids which might play a role in complexing phosphotyrosine by this antibody, a molecular model for the Py20 Fv was generated using the 3-D crystal structure coordinates of a closely related IgG (R19.9) and the molecular modeling programs CHARMm and CONGEN. The resulting model was tested by mutational analysis. Twelve amino acid residues in both the Py20 heavy and light chain variable regions were altered and the effects of these mutations on phosphotyrosine recognition were determined by affinity chromatography, a competitive ELISA and an antigen overlay assay. The results from this analysis revealed that light chain tryptophan 96 and heavy chain arginines 99 and 59, both of two tyrosines 105 and 106 and a histidine 35 were necessary for high affinity binding. Surprisingly, changing the heavy chain tyrosines 105 and 106 individually resulted in an FV that bound phosphotyrosine with an apparent affinity 10-fold greater than the wild type molecule. Even though the 105 and 106 FVs are monovalent, they recognize tyrosine-phosphorylated proteins displayed on a Western blot as well as the intact IgG. Five other amino acid changes had no obvious effect on antigen binding in any of the assays used.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.
authors.forename: S
authors.initials: S
authors.lastname: Ruff-Jamison
authors.forename: J R
authors.initials: JR
authors.lastname: Glenney
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013698:Templates, Genetic; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019000:Phosphotyrosine; D014443:Tyrosine