Conservation of binding site geometry among p-azophenylarsonate-specific antibodies.
Murine A/J anti-p-azophenylarsonate mAb that express a dominant cross-reactive Id are encoded by a single set of germ-line VH and VL region genes. The crystal structure of the Fab of antibody 36-71, which uses this canonical set of genes but is somatically mutated, was previously determined. An Fab 36-71:phenylarsonate complex was modeled, identifying amino acid side chains that were proposed as contact residues to hapten. The remarkable conservation of these residues among canonical anti-p-azophenylarsonate antibodies suggested that the overall binding site geometry was maintained among somatically mutated antiarsonate monoclonal antibodies. To test this hypothesis, we used the germ-line-encoded antibody 36-65 to construct mutant antibodies, using oligonucleotide-directed mutagenesis, which differed only at the putative H chain hapten-contacting residues, and measured their hapten binding. A framework residue at H chain position 47 involved in a hydrogen bond network with CDR residues was also mutated. Substitution of several amino acids at each position permitted evaluation of the stereochemical requirements for binding. The results indicate the importance of aromatic stacking of two H chain tyrosine residues against the phenyl ring of the hapten in maintaining affinity, as well as strict complementarity at H chain position 35. The results are consistent with the crystal model of the combining site, and provide further evidence for conservation of the three-dimensional binding site motif among antiarsonate antibodies that bear a dominant heritable ld.
Parhami-Seren
B
B
Department of Surgery, Massachusetts General Hospital, Boston 02114.
Kussie
P H
PH
Strong
R K
RK
Margolies
M N
MN
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D006801:Humans; D007130:Immunoglobulin Idiotypes / Q000032:analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011863:Radioimmunoassay; D013329:Structure-Activity Relationship; D010132:p-Azobenzenearsonate / Q000276:immunology*
D007130:Immunoglobulin Idiotypes; D010132:p-Azobenzenearsonate