Jeffrey P D, Schildbach J F, Chang C Y, Kussie P H, Margolies M N, Sheriff S
Department of Macromolecular Crystallography, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.
J Mol Biol. 1995 Apr 28;248(2):344-60. doi: 10.1016/s0022-2836(95)80055-7.
We determined the sequence, specificity for structurally related cardenolides, and three-dimensional structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain. The 40-50 antibody does not share close sequence homology with other high-affinity anti-digoxin antibodies. Measurement of the binding constants of structurally distinct digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40-50-ouabain Fab complex crystallizes in space group C2 with cell dimensions of a = 93.7 A, b = 84.8 A, c = 70.1 A, beta = 128.0 degrees. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 A. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the antibody and hapten. The surface area of the antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex allows qualitative explanation of the observed fine specificities of 40-50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab complexed with digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.
我们确定了抗地高辛抗体40 - 50 Fab与哇巴因复合物的序列、对结构相关强心甾类化合物的特异性以及三维结构。40 - 50抗体与其他高亲和力抗地高辛抗体没有密切的序列同源性。对结构不同的地高辛类似物结合常数的测量表明，其具有明确的特异性模式，也与其他抗地高辛抗体不同。40 - 50 -哇巴因Fab复合物在空间群C2中结晶，晶胞参数为a = 93.7 Å，b = 84.8 Å，c = 70.1 Å，β = 128.0°。通过X射线晶体学确定了复合物的结构，并在2.7 Å的分辨率下进行了精修。半抗原结合在一个从结合位点中心延伸穿过轻链可变结构域形成的沟槽样口袋中，六个互补决定区中的五个参与了与半抗原的相互作用。复合物中约四分之三的半抗原表面积被掩埋；抗体与半抗原之间形成了两个氢键。哇巴因掩埋的抗体结合位点表面积由轻链和重链可变结构域平均贡献。Fab上被掩埋的表面积超过一半由芳香族侧链组成。半抗原与抗体之间的表面互补性足以使复合物仅对半抗原中的一种内酯环构象具有特异性。40 - 50 -哇巴因复合物的晶体结构能够定性解释观察到的40 - 50的精细特异性，包括对在16和12位取代的半抗原的结合。将40 - 50与哇巴因复合的晶体结构与先前确定的26 - 10抗地高辛Fab与地高辛复合的晶体结构进行比较，表明这两种抗体以不同的方向结合这些结构相关的半抗原，这与其不同的精细特异性一致。这些结果表明，免疫系统能够产生抗体，为即使是小分子的结合提供多种结构解决方案。
