Structure, Specificity and Affinity Maturation of Anti-nitrophenol Antibody Mab 88C6/12: Fab Fragment and its Complex with a Peptide Analog Found from Peptide Library Search. Kon Ho Lee, Min Li, Mette Strand and L. Mario Amzel, Dept. of Physics, Dept. of Physiology, Dept. of Pharmacology, Dept. of Biophysics and Biophysical Chemistry School of Medicine, The Johns Hopkins University, Baltimore MD 21205

Crystal structures of the Fab fragment of an anti-nitrophenol(NP) antibody, MAb 88C6/12, and one of its complexes have been determined to resolution 2.8Å, 2.5Å to study affinity maturation and specificity of anitbodies. The complex contains one of the peptides found from a phage display random peptide library. It is known from a previous study of the Fab [epsilon]-amino-carproate NP complex that the nitro and hydroxyl groups of the hapten contribute to binding through interactions with residues W91,W96 in L3, H35 in H1, R50, K58 in H2, Y95,C98, R99 in H3. The structure of the uncomplexed Fab fragment of MAb 88C6/12 is similar to that of the NP complex except for the different conformation of CDR3 of the heavy chain(H3) and residue R50 in H2. H3 in Fab88C6/12 moves out about 1.68Å compared to that of the Fab-NP complex. Also the side chain of R50 in H2 moves into the binding pocket occupied by the NP head group in the Fab-NP complex. A phage display library was used to find peptide analogs which mimic the hapten, nitrophenol. Four types of clones have been identified from DNA sequences among 23 which showed high positive signal in ELISA. One of them, APWEQYTEL, was synthesized and cocrystallized in the same conditions. In the Fab-peptide complex structure, the H3 loop has the same conformation as that in the Fab structure, but the side chain of R50 moved away from the binding pocket. Four residues of the peptide, WEQY, were placed in the electron density. No significant density was found for other residues. It is thought that other residues of the peptide are hanging out from the binding pocket. The indol ring of the tryptophan of the peptide is anckored into the binding pocket surrounded by the residues, W91L3, W96L3, Y32L1, L33H1, H35H1, R50H2, Y95, and Y97. The tryptophan side chain of the peptide are stacking with that of two tryptophans from CDRs. Glu4 and Gln5 of the peptide participate in binding through hydrogen bonds with R50H2, K58H2.