From the Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China,; the Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, China.
the International Joint Cancer Institute, The Second Military Medical University, Shanghai 200433, China, and; the State Key Laboratory of Antibody Medicine and Targeting Therapy, Shanghai 201203, China.
J Biol Chem. 2013 Aug 30;288(35):25165-25172. doi: 10.1074/jbc.M113.480830. Epub 2013 Jul 12.
Ricin belongs to the type II ribosome-inactivating proteins that depurinate the universally conserved α-sarcin loop of rRNA. The RNA N-glycosidase activity of ricin also largely depends on the ribosomal proteins that play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface that contains both hydrogen bonds and van der Waals contacts. The complementarity-determining region loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues Asp(96)-Thr(116)). ELISA results show that Gln(98), Glu(99), Glu(102), and Thr(105) (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome and the surface plasmon resonance and pulldown results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity.
蓖麻毒素属于 II 型核糖体失活蛋白，可使 rRNA 普遍保守的 α-兴孢菌素环脱嘌呤。蓖麻毒素的 RNA N-糖苷酶活性也在很大程度上依赖于核糖体蛋白，这些蛋白在 rRNA 脱嘌呤过程中发挥重要作用。因此，研究蓖麻毒素与核糖体元件的相互作用将有助于更好地理解蓖麻毒素的催化机制。抗体 6C2 是一种具有异常强大中和能力的鼠单克隆抗体，可中和蓖麻毒素，但中和机制仍不清楚。在这里，我们报告了 6C2 Fab 与蓖麻毒素 A 链（RTA）复合物的 2.8Å 晶体结构，该结构揭示了一个广泛的抗原-抗体界面，包含氢键和范德华接触。互补决定区环 H1、H2、H3 和 L3 形成一个口袋，以容纳 RTA 上的表位（残基 Asp（96）-Thr（116））。ELISA 结果表明，RTA 上的 Gln（98）、Glu（99）、Glu（102）和 Thr（105）是识别 6C2 的关键残基。通过干扰 6C2 Fab-RTA 界面，6C2 失去了中和能力，这可以通过在无细胞系统中抑制蛋白质合成来衡量。最后，我们提出 6C2 中和蓖麻毒素的机制是，6C2 的结合阻碍了 RTA 与核糖体的相互作用，表面等离子体共振和下拉结果证实了我们的假设。总之，我们的数据解释了 mAb 6C2 中和蓖麻毒素的机制，并为开发具有更好特异性和更高亲和力的改良抗体药物提供了结构基础。
