A mutational analysis of the binding of two different proteins to the same antibody.
The crystal structures of the complexes between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL and between D1.3 and the anti-D1.3 antibody E5.2 have shown that D1.3 contacts these two proteins through essentially the same set of combining site residues [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., & Mariuzza, R. A. (1995) Nature 374, 739-742]. To probe the relative contribution of individual residues to complex stabilization, single alanine substitutions were introduced in the combining site of D1.3, and their effects on affinity for HEL and for E5.2 were measured using surface plasmon resonance detection, fluorescence quench titration, or sedimentation equilibrium. The energetics of the binding to HEL are dominated by only 3 of the 13 contact residues tested (delta Gmutant-delta Gwild type > 2.5 kcal/mol): VLW92, VHD100, and VHY101. These form a patch at the center of the interface and are surrounded by residues whose apparent contributions are much less pronounced ( < 1.5 kcal/mol). This contrasts with the interaction of D1.3 with E5.2 in which most the contact residues (11 of 15) were found to play a significant role in ligand binding ( > 1.5 kcal/mol). Furthermore, even though D1.3 contacts HEL and E5.2 in very similar ways, the functionally important residues of D1.3 are different for the two interactions, with only substitutions at D1.3 positions VH100 and VH101 greatly affecting binding to both ligands. Thus, the same protein may recognize different ligands in ways that are structurally similar yet energetically distinct.
Dall'Acqua
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Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Goldman
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Eisenstein
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Mariuzza
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002645:Chickens; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005260:Female; D005803:Genes, Immunoglobulin; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D000906:Antibodies; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D009113:Muramidase