Nanobody Inhibitors Target the Coiled Coil Arms of the Bacillus subtilis SMC complex

Synthetic nanobodies—also called sybodies—have proven valuable for stabilizing conformations of purified proteins, advancing structural and functional studies for example of transmembrane protein complexes. However, their utility in modulating protein function in living cells has remained less well explored. Structural Maintenance of Chromosomes (SMC) complexes facilitate chromosome organization by DNA loop extrusion, a fundamental process in all domains of life. In this study, we target the bacterial SMC complex, Smc-ScpAB, in Bacillus subtilis with synthetic nanobodies, with the aim to identify key functional regions of the protein complex in a largely unbiased manner. Using a previously established high-throughput selection platform, we first isolate nanobodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select nanobodies capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the fourteen disruptive nanobodies belong to one of three library designs, target the Smc subunit near the same coiled coil arm interface and modulate its ATPase activity in two principal ways, highlighting the mid-region of Smc coiled coil as critical feature of the DNA folding process. These findings underscore the potential of nanobodies—and, by extension, designed binders—as versatile tools for probing dynamic protein function in living cells, with potentially broad applications in cell and synthetic biology.

合成纳米抗体（synthetic nanobodies，亦称sybodies）已被证实可有效稳定纯化蛋白的构象，有力推动了跨膜蛋白复合物等的结构与功能研究。然而，其在活细胞内调控蛋白功能的应用潜力尚未得到充分探索。染色体结构维持复合物（Structural Maintenance of Chromosomes, SMC）通过DNA环挤出过程介导染色体组织，这是所有生命域中的基础生理过程。本研究以枯草芽孢杆菌（Bacillus subtilis，下文简称B. subtilis）中的细菌SMC复合物Smc-ScpAB为研究对象，采用合成纳米抗体开展实验，旨在以近乎无偏的方式鉴定该蛋白复合物的关键功能区域。借助此前已建立的高通量筛选平台，我们首先分离出可特异性结合纯化Smc-ScpAB的纳米抗体，随后在枯草芽孢杆菌中表达这些抗体，筛选出能够破坏Smc-ScpAB功能的纳米抗体，此类抗体可引发染色体分离缺陷与细胞死亡。作图分析与生化表征结果显示，这14株具有破坏活性的纳米抗体分属3种文库设计之一，均靶向Smc亚基的同一卷曲螺旋臂结合界面，并通过两种主要方式调控其ATP酶（ATPase）活性，这凸显了Smc卷曲螺旋的中段区域是DNA折叠过程的关键特征位点。本研究结果证实了纳米抗体——以及由此推广的定制化结合蛋白——作为通用工具的潜力，可用于探究活细胞内动态的蛋白功能，在细胞生物学与合成生物学领域具备广阔的应用前景。