Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody.
The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr(32)Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe(94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe(94)Ala up to the level of the e-pep affinity to the wild-type scFv.
Winkler
K
K
Institute of Biochemistry and Institute of Medical Immunology, Medical Department, Humboldt University, Berlin, Germany.
Kramer
A
A
Küttner
G
G
Seifert
M
M
Scholz
C
C
Wessner
H
H
Schneider-Mergener
J
J
Höhne
W
W
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics*; D001667:Binding, Competitive / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D015483:HIV Antibodies / Q000235:genetics / Q000378:metabolism*; D016655:HIV Core Protein p24 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D017354:Point Mutation / Q000276:immunology*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; C055966:immunoglobulin Fv; D010649:Phenylalanine; D000409:Alanine