Single Domain Antibody 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. We first isolate sybodies that specifically bind purified Smc-ScpAB and then express them in B. subtilis to select binders capable of disrupting Smc-ScpAB function, leading to chromosome segregation defects and cell death. Mapping and biochemical characterization show that the fourteen disruptive sybodies 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 sybodies—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，为跨膜蛋白复合物等的结构与功能研究提供了重要助力。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核心基础生物学过程。本研究以枯草芽孢杆菌（Bacillus subtilis）中的细菌SMC复合物Smc-ScpAB为研究对象，使用合成纳米抗体，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。我们首先分离出可特异性结合纯化Smc-ScpAB的sybodies，随后在枯草芽孢杆菌中表达这些抗体，以筛选能够破坏Smc-ScpAB功能的结合剂，最终导致染色体分离缺陷与细胞死亡。图谱分析与生化表征结果显示，这14株具有破坏功能的sybodies分属于3种文库设计之一，均靶向Smc亚基靠近同一卷曲螺旋臂界面的区域，并通过两种主要方式调控其ATP酶活性，证实Smc卷曲螺旋的中部区域是DNA折叠过程的关键特征位点。本研究结果凸显了sybodies——以及由此推广的定制化结合剂——作为探究活细胞内动态蛋白功能的通用工具的潜力，在细胞生物学与合成生物学领域具备广阔的应用前景。