Characterization of an anti-digoxin antibody binding site by site-directed in vitro mutagenesis.
In vitro mutagenesis and immunoglobulin gene transfection were used to investigate the binding site of a monoclonal antibody, 2610, that binds to digoxin, a cardiac glycoside. A computer model was generated in order to select sites in the complementarity determining regions (CDR) that would participate in binding. Residues in the CDR segments were chosen that possess high solvent exposure and were located in a putative cleft. The cloned heavy and light chain variable regions were subjected to in vitro mutagenesis at these sites. The mutated variable regions in M13 were then subcloned into expression vectors and transfected. The affinities and specificity binding properties of the resultant expressed antibodies were measured. Many of the mutants of the putative contact residues showed significant but not major alterations of binding properties. Since most of the residues in the binding site are non-polar and aromatic and since many of the mutations resulted in only modest binding changes, we theorize that much of the high affinity binding (> 10(9)/M) is the cumulation of many weak interactions, arising from dispersion forces and hydrophobic effects in the pocket. Preliminary mutagenesis of two L chain positions proposed to bind to the lactone end of digoxin have larger binding effects. Specificity studies show that the mutants more frequently possess altered binding to the lactone ring of digoxin that altered binding to other digoxin moieties. The data are most suggestive of a model in which lactone is at the bottom of a binding pocket, followed by the steroid nucleus and then by the sugar moiety extruding out of the pocket. The binding information may be useful in understanding the immune response to large, hydrophobic haptens.
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Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston 02114.
Mudgett-Hunter
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Novotny
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Bruccoleri
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity / Q000235:genetics; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody / Q000235:genetics* / Q000502:physiology; D003001:Cloning, Molecular; D003198:Computer Simulation; D004077:Digoxin / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D004077:Digoxin