Probing antibody diversity by 2D NMR: comparison of amino acid sequences, predicted structures, and observed antibody-antigen interactions in complexes of two antipeptide antibodies.
The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specificities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.
Levy
R
R
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
Assulin
O
O
Scherf
T
T
Levitt
M
M
Anglister
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000916:Antibody Diversity; D000937:Antigen-Antibody Reactions; D002772:Cholera Toxin / Q000276:immunology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D012689:Sequence Homology, Nucleic Acid; D013057:Spectrum Analysis; D014443:Tyrosine
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D014443:Tyrosine; D006639:Histidine; C050310:cholera toxin, B subunit (50-64); D002772:Cholera Toxin