RESUMEN
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.
Asunto(s)
Sustitución de Aminoácidos/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Muramidasa/inmunología , Mutagénesis Sitio-Dirigida , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión de Anticuerpos/genética , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Cristalografía por Rayos X , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Lisina/genética , Sustancias Macromoleculares , Ratones , Muramidasa/química , Fenilalanina/genética , Conformación Proteica , Serina/genética , Valina/genéticaRESUMEN
The crystal structure of the complex between hen egg lysozyme and the Fv fragment of a humanized antilysozyme antibody was determined to 2.7-A resolution. The structure of the antigen combining site in the complex is nearly identical to that of the complexed form of the parent mouse antibody, D1.3. In contrast, the combining sites of the unliganded mouse and humanized antilysozymes show moderate conformational differences. This disparity suggests that a conformational readjustment process linked to antigen binding reverses adverse conformations in the complementarity determining regions that had been introduced by engineering these segments next to human framework regions in the humanized antibody.