| The stereoselective interaction between proteins and low molecular weight ligands, such as drugs, was first described more than a century ago, yet the molecular basis of chiral biodiscrimination remains poorly understood. In this study, the interaction between monoclonal antibodies and amino acid enantiomers was investigated as a model system to better understand the molecular basis of chiral discrimination mediated by protein binding sites.;The antibodies investigated here had previously been produced by immunizing mice with conjugates of either p-amino-D- or -L-phenylalanine and keyhole limpet hemocyanin (KLH), synthesized by diazotization. The fusion of antibody-producing B-lymphocytes with myeloma cells yielded hybridoma cells that were expanded in this work for mRNA isolation and subsequent sequencing. A detailed analysis of sequences of various anti-D- and anti-L-amino acid antibodies as well as some stereoselective anti-hydroxy acid antibodies indicated that most of the investigated immunoglobulins were sequentially very similar and derived from the same germline progenitor. Interestingly, this finding was independent of class specificity or enantioselectivity displayed by the antibodies.;To further elucidate factors governing antibody stereoselectivity at the atomic level, molecular modeling and ligand docking studies were performed on the binding sites of two anti-amino acid antibodies that exhibit opposing enantioselectivity but have highly homologous amino acid sequences. It was found that both immunoglobulins bind their ligands through hydrophobic interactions and a network of hydrogen bonds that involve the substituents about the ligand's stereogenic center. For both antibodies, rejection of the "wrong" enantiomer of the ligand is based on steric hindrance.;Since the primary structures of these two antibodies differ only by a few residues, it was possible to identify those positions that are decisive for determining whether the antibodies' binding sites recognize either D- or L-amino acid ligands. In silico mutation of those residues resulted in the conversion of an anti-L-amino acid antibody into an anti-D-amino acid antibody. Docking studies performed with this converted antibody structure showed that its binding affinity for D-amino acids was comparable to that of the original anti-D-amino acid antibody.;Finally, molecular modeling was utilized to obtain the structure of the germline progenitor, which was used in docking studies. While attempts to dock either enantiomer of phenylalanine to this structure failed, the incorporation of mutations common to both matured stereoselective antibodies resulted in an antibody capable of binding D- and L-phenylalanine. |
