Energetic and kinetic contributions of contact residues of antibody D1.3 in the interaction with lysozyme.
Fully functional variable fragments (Fv) of D1.3, a mouse antibody directed against the hen egg lysozyme, were readily produced as hybrids (Fv-MalE) with the maltose-binding protein of Escherichia coli and purified independently of their antigen-binding properties. We used site-directed mutations of residues in the complementarity-determining regions (CDRs) of D1.3 as local conformational probes, and compared their effects on the binding of Fv and Fv-MalE to lysozyme. We found that the MalE moiety did not significantly interfere with the interaction between the antigen and the antibody Fv fragment. We then determined the contribution of several potential contact residues of D1.3 in the interaction with lysozyme, by assaying the effect of site-directed mutations on the kinetics of association and dissociation of the complex between Fv-MalE and immobilized lysozyme, using the BIAcore apparatus. While the k(on) values were virtually unaffected by the mutations, the k(off) values varied by more than three orders of magnitude. Both charged (aspartate and arginine) and aromatic (tyrosine and tryptophan) residues in the CDR3 regions of the heavy and light chains of D1.3, which form the center of its antigen-combining site, played a preponderant part in the binding of lysozyme. Our results also showed that indirect hydrogen bonds, bridged by water molecules, contributed significantly to the interaction between D1.3 and lysozyme, and that their energy could be estimated at 1 to 2 kcal.mol-1.
England
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Protein-Engineering Group (CNRS-URA 1129), Institut Pasteur, Paris, France.
Brégégère
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Bedouelle
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D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000276:immunology / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics*; D002645:Chickens; D018360:Crystallography, X-Ray; D004527:Egg Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D058619:Maltose-Binding Proteins; D051379:Mice; D009004:Monosaccharide Transport Proteins; D009113:Muramidase / Q000276:immunology*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins; D004527:Egg Proteins; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D009113:Muramidase