Three-dimensional Structures of the Fab Fragment of Murine N1G9 Antibody from the Primary Immune Response and of its Complex with (4-Hydroxy-3-Nitrophenyl)acetate

Abstract

The three-dimensional structures of the Fab fragment, in its unliganded and liganded crystals, of mouse anti-(4-hydroxy-3-nitrophenyl)acetate (NP) antibody N1G9 have been determined by the molecular replacement method. The unliganded and NP-liganded structures were refined at 2.4 Å resolution to crystallographic R-factors of 0.194 and 0.196, respectively. Antibody N1G9 bears |lclambd| light chains, and is one of the primary immune response antibodies. Fab N1G9 exhibits an elbow angle of 197° in both structures. This large angle is ascribed to the V L–C Linterface formed by |lclambd|- chain residues. A hydrophobic pocket surrounded by the complementarity- determining regions except L2 is identified as a hapten-binding site. Be- tween the liganded and unliganded structures, root-mean-square (r.m.s.) positional deviations are 0.42 Å for the main-chain atoms, and 0.74 Å for all the protein atoms. The major structural differences between these structures are localized in the hapten-binding site, and yield an r.m.s. deviation of 1.03 Å for the side-chain atoms. The soaked NP ligand is in van der Waals contact with the aromatic side-chains of Tyr32L and Trp91L of the light chain, and Trp33H and Tyr97H of the heavy chain, and is hydrogen-bonded to the side-chains of Trp96L, His35H, Arg50H, Tyr95H, and Ser100aH. The side-chain of Lys58H is salt-bridged to the NP hydroxyl group. The side-chains of Arg50H, Trp33H, and Tyr97H are shifted toward the NP carboxyl group. The side-chain of Trp33H, whose replacement to Leu increases affinity by tenfold, is sandwiched between the Arg50H and Tyr97H side-chains, and is in cramped contact both with the ligand and with these side-chains. Affinity increases in the maturation of the anti-NP antibodies are ascribable to conformational relief of these cramped contacts through the replacement of Trp33H or through suitable structural alterations in the H3 region.