Structural effects of framework mutations on a humanized anti-lysozyme antibody

A humanized version of the mouse anti-lysozyme Ab D1.3 was previously const ructed as an Fv fragment and its structure was crystallographically determi ned in the free form and in complex with lysozyme. Here we report five new crystal structures of single-amino acid substitution mutants of the humaniz ed 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 Angstrom. Residue 27 in other Abs has been implicated in stabilizing the conformation of the first compl ementarity-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 ad jacent residues, but this change is only weakly transmitted to Ag binding r esidues in the nearby CDR. Residue 71 of the H chain has been proposed to c ontrol 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 A rg substituted in place of Lys at position 71, no significant change in the conformation of CDRs 1 and 2 was observed.