Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4.
Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4; Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
J Mol Biol. 2018 Feb 2;430(3):322-336. doi: 10.1016/j.jmb.2017.12.010. Epub 2017 Dec 22.
Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody-antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (VH) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody-ligand complex and lowers its aggregation temperature to favor crystallization. The VH crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules.
单克隆抗体是治疗癌症和免疫紊乱的最大药物类别之一，并且正在指导针对传染病的疫苗设计。由于相对较低的灵活性，片段抗原结合（Fabs）优先于单克隆抗体用于抗体-抗原复合物的结构特征描述。尽管如此，由于互补决定区的表面特征和可变域和恒定域之间铰链区的剩余灵活性，Fabs 仍然常常难以结晶。在这里，我们使用了针对人κ轻链的可变重链（VH）结构域来辅助自身难以结晶的三种治疗性 Fab 的结构确定。我们表明，这种配体改变了抗体-配体复合物的表面特性，并降低了其聚集温度以有利于结晶。VH 结晶伴侣还将 Fab 的柔性铰链限制在一个狭窄的角度范围内，并且独立于可变区。我们的发现为抗体结构确定提供了有价值的方法，并为控制大分子结晶的原则提供了生物物理见解。
