Essen L O, Skerra A
Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
J Mol Biol. 1994 Apr 29;238(2):226-44. doi: 10.1006/jmbi.1994.1284.
In a protein design approach the molecular model of an artificial antibody Fv fragment was generated with predicted complementarity to part of the known crystal structure of chicken egg-white cystatin. The model of the Fv fragment was based on the three-dimensional structure of the anti-lysozyme antibody HyHEL-10, which was modified by substituting amino acid side-chains in the complementarity-determining regions (CDRs) as well as the framework without altering the backbone. In the course of crystallization experiments with the bacterially produced Fv fragment crystals of the VL domain alone were obtained. These crystals diffracted X-rays to a resolution of 2.17 A and were shown to belong to the space group P2(1)2(1)2(1) with unit cell dimensions a = 46.89 A, b = 58.05 A, c = 83.22 A containing two VL monomers in the asymmetric unit. The crystal structure was solved by molecular replacement and refined to a crystallographic R-factor of 17.5%. The two VL monomers exhibit an asymmetric mode of association, which is different from other crystallized VL domains described before and shows the peculiar feature of an isopropanol precipitant molecule buried at the interface. Both VL structures reveal a high level of similarity to the predicted three-dimensional model. With the exception of two loop segments in the framework region that are involved in crystal packing contacts, the backbone structures of the two VL monomers in the crystal and the molecular model of the VL domain are practically identical. Although six amino acid residues had been replaced in the hypervariable regions, the CDR conformations remained conserved and only minor deviations in the orientation of some side-chains and peptide planes were detected. The crystallographic analysis of the VL domain modelled as part of a complex between an artificial Fv fragment and the small protein cystatin, deliberately chosen as antigen target, confirms the concept of distinct structural classes for CDR backbones and supports our strategy for the de novo design of an antibody combining site.
在一种蛋白质设计方法中，生成了人工抗体Fv片段的分子模型，该模型与鸡卵清半胱氨酸蛋白酶抑制剂已知晶体结构的一部分具有预测的互补性。Fv片段的模型基于抗溶菌酶抗体HyHEL-10的三维结构，通过在互补决定区（CDR）以及框架中替换氨基酸侧链进行修饰，而不改变主链。在用细菌产生的Fv片段进行结晶实验过程中，仅获得了VL结构域的晶体。这些晶体将X射线衍射至2.17 Å的分辨率，显示属于空间群P2(1)2(1)2(1)，晶胞尺寸a = 46.89 Å，b = 58.05 Å，c = 83.22 Å，不对称单位中包含两个VL单体。通过分子置换解析了晶体结构，并将其精修至晶体学R因子为17.5%。两个VL单体呈现出不对称的缔合模式，这与之前描述的其他结晶VL结构域不同，并且显示出在界面处埋入异丙醇沉淀剂分子的独特特征。两种VL结构都与预测的三维模型高度相似。除了框架区域中参与晶体堆积接触的两个环段外，晶体中两个VL单体的主链结构与VL结构域的分子模型实际上是相同的。尽管在高变区有六个氨基酸残基被替换，但CDR构象仍然保守，仅检测到一些侧链和肽平面方向的微小偏差。对作为人工Fv片段与特意选为抗原靶标的小蛋白质半胱氨酸蛋白酶抑制剂之间复合物一部分建模的VL结构域进行晶体学分析，证实了CDR主链存在不同结构类别的概念，并支持了我们从头设计抗体结合位点的策略。
