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Structural effects of framework mutations on a humanized anti-lysozyme antibody
M A Holmes1, T N Buss, J Foote
1Program in Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Journal of Immunology (Baltimore, Md. : 1950)
|June 22, 2001
Summary
Structural analysis of a humanized antibody fragment (Fv) revealed that mutations at residue 27 only weakly affect antigen binding sites. Substitutions at residue 71 did not alter complementarity-determining region conformations.
Area of Science:
- Structural Biology
- Immunology
- Protein Engineering
Background:
- A humanized anti-lysozyme antibody fragment (Fv) D1.3 was previously characterized structurally.
- Understanding antibody structure-function relationships is crucial for therapeutic antibody development.
Purpose of the Study:
- To investigate the structural impact of specific amino acid substitutions in a humanized Fv fragment.
- To determine how mutations at residues 27 and 71 affect antibody fragment conformation and antigen binding.
Main Methods:
- X-ray crystallography was used to determine the structures of five single-amino acid substitution mutants of the humanized Fv fragment.
- Four mutant structures were determined in complex with lysozyme.
- Structures were refined to resolutions of 2.7-2.9 Å.
Main Results:
- A Phe-to-Ser mutation at residue 27 altered local conformations but had minimal impact on antigen binding residues in the nearby complementarity-determining region (CDR).
- Substitutions of Lys at residue 71 with Val, Ala, or Arg did not significantly alter the conformation of H chain CDRs 1 and 2.
Conclusions:
- Residue 27 plays a limited role in stabilizing the overall conformation of the H chain CDRs involved in antigen binding.
- The side chain bulk of residue 71 does not appear to control the relative disposition of H chain CDRs 1 and 2.