Functional mapping of conserved residues located at the VL and VH domain interface of a Fab.
The interface between the VL and VH domains of antibodies is highly conserved. To investigate the influence of conserved interface residues on Fab function, 13 interface residues were subjected to codon-based combinatorial alanine scanning mutagenesis in Fab 57P, specific for peptide 134 to 151 of the coat protein of tobacco mosaic virus. The 13 single mutants were analysed by Western blot to determine the effect of interface modifications on Fab expression. The kinetic rate constants of peptide-Fab mutant interactions were measured using the biosensor technology. Alanine replacements did not prevent assembly of the mutated Fabs and led to a modification of their binding properties in every case. Twelve of the 13 target residues correspond to homologous positions in the VL and VH domains, which have similar folds. Mutation at homologous positions mostly had different effects on antigen binding affinity. The replacement of bulky side-chains had the most drastic effect on binding. When smaller side-chains were replaced by alanine, the binding properties of Fab mutants differed slightly (by less than a factor of two), but significantly from that of Fab 57P. Modification of some of these residues, which are located 9 to 12 A away from the base of CDR loops, is unlikely to alter loop conformation. They may affect antigen binding indirectly by influencing the relative position of the VL and VH domains. Our results demonstrate that residues situated at the VL-VH interface and which are remote from the paratope are able to influence the antigen binding properties of antibodies.
Chatellier
J
J
Institut de Biologie Moléculaire et Cellulaire (CNRS), Laboratoire d'Immunochimie des Peptides et des Virus, Stransbourg, France.
Van Regenmortel
M H
MH
Vernet
T
T
Altschuh
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002213:Capsid / Q000276:immunology; D017124:Conserved Sequence; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000276:immunology; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D014027:Tobacco Mosaic Virus / Q000276:immunology
D000914:Antibodies, Viral; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments