Mizutani R, Miura K, Nakayama T, Shimada I, Arata Y, Satow Y
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
J Mol Biol. 1995 Nov 24;254(2):208-22. doi: 10.1006/jmbi.1995.0612.
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 A resolution to crystallographic R-factors of 0.194 and 0.196, respectively. Antibody N1G9 bears lambda light chains, and is one of the primary immune response antibodies. Fab N1G9 exhibits an elbow angle of 197 degrees in both structures. This large angle is ascribed to the VL-CL interface formed by lambda-chain residues. A hydrophobic pocket surrounded by the complementarity-determining regions except L2 is identified as a hapten-binding site. Between the liganded and unliganded structures, root-mean-square (r.m.s.) positional deviations are 0.42 A for the main-chain atoms, and 0.74 A 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 A 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.
通过分子置换法测定了小鼠抗（4-羟基-3-硝基苯基）乙酸酯（NP）抗体N1G9的Fab片段在未结合配体和结合配体晶体中的三维结构。未结合配体和结合NP配体的结构分别在2.4埃分辨率下进行精修，晶体学R因子分别为0.194和0.196。抗体N1G9带有λ轻链，是主要免疫反应抗体之一。Fab N1G9在两种结构中均呈现197度的肘角。这个大角度归因于由λ链残基形成的VL-CL界面。除L2外，由互补决定区包围的一个疏水口袋被确定为半抗原结合位点。在结合配体和未结合配体的结构之间，主链原子的均方根（r.m.s.）位置偏差为0.42埃，所有蛋白质原子的偏差为0.74埃。这些结构之间的主要结构差异位于半抗原结合位点，侧链原子的r.m.s.偏差为1.03埃。浸泡的NP配体与轻链的Tyr32L和Trp91L以及重链的Trp33H和Tyr97H的芳香侧链存在范德华接触，并与Trp96L、His35H、Arg50H、Tyr95H和Ser100aH的侧链形成氢键。Lys58H的侧链与NP羟基形成盐桥。Arg50H、Trp33H和Tyr97H的侧链向NP羧基移动。Trp33H的侧链被Leu取代后亲和力提高了十倍，它夹在Arg50H和Tyr97H侧链之间，与配体以及这些侧链都紧密接触。抗NP抗体成熟过程中亲和力的增加归因于通过Trp33H的取代或H3区域的适当结构改变来缓解这些紧密接触的构象。
