title: Structural insights into the neutralization mechanism of monoclonal antibody 6C2 against ricin.
abstract: 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.
authors.affiliation: 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.
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: 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.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Dai
authors.affiliation: 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.
authors.forename: Tiancheng
authors.initials: T
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Fang
authors.affiliation: 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.
authors.forename: Huajing
authors.initials: H
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: 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.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Yu
authors.affiliation: 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.
authors.forename: Tian
authors.initials: T
authors.lastname: Xia
authors.affiliation: 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.
authors.forename: Liwen
authors.initials: L
authors.lastname: Niu
authors.affiliation: 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. Electronic address: yjguo@smmu.edu.cn.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Guo
authors.affiliation: 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,. Electronic address: mkteng@ustc.edu.cn.
authors.forename: Maikun
authors.initials: M
authors.lastname: Teng
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000378:metabolism; D001666:Binding Sites, Antibody; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012276:Ricin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D057134:Antibodies, Neutralizing; D022801:Complementarity Determining Regions; D012276:Ricin
keywords: Antibodies; RIP; Ribosomal RNA Processing; Ribosomes; Structural Biology