Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA; GSK, Rockville, MD 20850, USA.
Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA.
Structure. 2021 Jun 3;29(6):598-605.e3. doi: 10.1016/j.str.2021.02.001. Epub 2021 Feb 25.
This work presents a method for introducing synthetic symmetry into protein crystallization samples using an antibody fragment termed a diabody (Dab). These Dabs contain two target binding sites, and engineered disulfide bonds have been included to modulate Dab flexibility. The impacts of Dab engineering have been observed through assessment of thermal stability, small-angle X-ray scattering, and high-resolution crystal structures. Complexes between the engineered Dabs and HIV-1 reverse transcriptase (RT) bound to a high-affinity DNA aptamer were also generated to explore the capacity of engineered Dabs to enable the crystallization of bound target proteins. This strategy increased the crystallization hit frequency obtained for RT-aptamer, and the structure of a Dab-RT-aptamer complex was determined to 3.0-Å resolution. Introduction of synthetic symmetry using a Dab could be a broadly applicable strategy, especially when monoclonal antibodies for a target have previously been identified.
这项工作提出了一种在蛋白质结晶样品中引入合成对称的方法，使用一种称为二抗体（Dab）的抗体片段。这些二抗包含两个靶结合位点，并设计了二硫键以调节二抗的灵活性。通过评估热稳定性、小角 X 射线散射和高分辨率晶体结构来观察二抗工程的影响。还生成了工程化二抗与与高亲和力 DNA 适体结合的 HIV-1 逆转录酶（RT）之间的复合物，以探索工程化二抗使结合靶蛋白结晶的能力。该策略增加了 RT-适体获得的结晶命中率，并且确定了 Dab-RT-适体复合物的结构分辨率为 3.0-Å。使用二抗引入合成对称可能是一种广泛适用的策略，特别是当已经鉴定出针对目标的单克隆抗体时。
