Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
ImmuneOnco Biopharmaceuticals (Shanghai) Co., Ltd., Shanghai 201203, China.
Molecules. 2022 Aug 30;27(17):5574. doi: 10.3390/molecules27175574.
Targeting the CD47/SIRPα signaling pathway represents a novel approach to enhance anti-tumor immunity. However, the crystal structure of the CD47/SIRPα has not been fully studied. This study aims to analyze the structure interface of the complex of CD47 and IMM01, a novel recombinant SIRPα-Fc fusion protein. IMM01-Fab/CD47 complex was crystalized, and diffraction images were collected. The complex structure was determined by molecular replacement using the program PHASER with the CD47-SIRPαv2 structure (PDB code 2JJT) as a search model. The model was manually built using the COOT program and refined using TLS parameters in REFMAC from the CCP4 program suite. Crystallization and structure determination analysis of the interface of IMM01/CD47 structure demonstrated CD47 surface buried by IMM01. Comparison with the literature structure (PDB ID 2JJT) showed that the interactions of IMM01/CD47 structure are the same. All the hydrogen bonds that appear in the literature structure are also present in the IMM01/CD47 structure. These common hydrogen bonds are stable under different crystal packing styles, suggesting that these hydrogen bonds are important for protein binding. In the structure of human CD47 in complex with human SIRPα, except SER66, the amino acids that form hydrogen bonds are all conserved. Furthermore, comparing with the structure of PDB ID 2JJT, the salt bridge interaction from IMM01/CD47 structure are very similar, except the salt bridge bond between LYS53 in IMM01 and GLU106 in CD47, which only occurs between the B and D chains. However, as the side chain conformation of LYS53 in chain A is slightly different, the salt bridge bond is absent between the A and C chains. At this site between chain A and chain C, there are a salt bridge bond between LYS53 (A) and GLU104 (C) and a salt bridge bond between HIS56 (A) and GLU106 (C) instead. According to the sequence alignment results of SIRPα, SIRPβ and SIRPγ in the literature of PDB ID 2JJT, except ASP100, the amino acids that form common salt bridge bonds are all conserved. Our data demonstrated crystal structure of the IMM01/CD47 complex and provides a structural basis for the structural binding interface and future clinical applications.
靶向 CD47/SIRPα 信号通路代表了增强抗肿瘤免疫的新方法。然而，CD47/SIRPα 的晶体结构尚未得到充分研究。本研究旨在分析 CD47 与新型重组 SIRPα-Fc 融合蛋白 IMM01 的复合物的结构界面。使用 PHASER 程序的分子置换方法，以 CD47-SIRPαv2 结构（PDB 代码 2JJT）作为搜索模型，对 IMM01-Fab/CD47 复合物进行了结晶和衍射图像收集。使用 COOT 程序手动构建模型，并使用 CCP4 程序套件中的 REFMAC 中的 TLS 参数进行精修。对 IMM01/CD47 结构界面的结晶和结构测定分析表明，CD47 表面被 IMM01 覆盖。与文献结构（PDB ID 2JJT）的比较表明，IMM01/CD47 结构的相互作用相同。文献结构中出现的所有氢键也存在于 IMM01/CD47 结构中。这些共同的氢键在不同的晶体包装方式下都很稳定，表明这些氢键对蛋白质结合很重要。在人 CD47 与人 SIRPα 复合物的结构中，除 SER66 外，形成氢键的氨基酸均保守。此外，与 PDB ID 2JJT 的结构相比，除了 IMM01/CD47 结构中 B 链和 D 链之间的盐桥相互作用外，其他盐桥相互作用非常相似，而 LYS53 与 CD47 中的 GLU106 之间的盐桥键仅存在于 B 链和 D 链之间。然而，由于 A 链中 LYS53 的侧链构象略有不同，A 链和 C 链之间不存在盐桥键。在 A 链和 C 链之间的这个位点，有一个 LYS53（A）与 GLU104（C）之间的盐桥键和一个 HIS56（A）与 GLU106（C）之间的盐桥键代替。根据文献中 PDB ID 2JJT 的 SIRPα、SIRPβ 和 SIRPγ 的序列比对结果，除 ASP100 外，形成共同盐桥键的氨基酸均保守。
我们的数据证明了 IMM01/CD47 复合物的晶体结构，为结构结合界面和未来的临床应用提供了结构基础。
