Structural effects of framework mutations on a humanized anti-lysozyme antibody.
A humanized version of the mouse anti-lysozyme Ab D1.3 was previously constructed as an Fv fragment and its structure was crystallographically determined in the free form and in complex with lysozyme. Here we report five new crystal structures of single-amino acid substitution mutants of the humanized Fv fragment, four of which were determined as Fv-lysozyme complexes. The crystals were isomorphous with the parent forms, and were refined to free R values of 28-31% at resolutions of 2.7-2.9 A. Residue 27 in other Abs has been implicated in stabilizing the conformation of the first complementarity-determining region (CDR) of the H chain, residues 31-35. We find that a Phe-to-Ser mutation at 27 alters the conformation of immediately adjacent residues, but this change is only weakly transmitted to Ag binding residues in the nearby CDR. Residue 71 of the H chain has been proposed to control the relative disposition of H chain CDRs 1 and 2, based on the bulk of its side chain. However, in structures we determined with Val, Ala, or Arg substituted in place of Lys at position 71, no significant change in the conformation of CDRs 1 and 2 was observed.
Holmes
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MA
Program in Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Buss
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TN
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D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology*; D000818:Animals; D001666:Binding Sites, Antibody / Q000235:genetics; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics*; D008239:Lysine / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D009113:Muramidase / Q000737:chemistry / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D012694:Serine / Q000235:genetics; D014633:Valine / Q000235:genetics
D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D012694:Serine; D010649:Phenylalanine; D009113:Muramidase; D014633:Valine; D008239:Lysine