Structural Biology Laboratory, G. N. Ramachandran Protein Centre, Council of Scientific and Industrial Research-Institute of Microbial Technology (CSIR-IMTECH), Chandigarh, India.
Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR-Biotech Science Cluster, Faridabad, India.
FEBS J. 2017 Dec;284(23):4066-4082. doi: 10.1111/febs.14289. Epub 2017 Oct 29.
VapBCs, virulence-associated proteins, are the most abundant type II toxin-antitoxin (TA) systems in prokaryotes. Under normal conditions, toxin and antitoxin interact to form a heterooctameric complex, which upon binding to operator sites, inhibits their own expression. Under stress conditions, the VapB antitoxin is degraded by cellular proteases to release a free VapC toxin, which in turn inhibits cell growth mainly by targeting protein translation. However, the intermediate steps involved in the assembly of the heterooctameric complex have not been resolved. Here, we report a 1.75 Å resolution crystal structure of VapC20, a Sarcin-Ricin loop cleaving toxin from type II TA system of Mycobacterium tuberculosis. Using analytical ultracentrifugation (AUC) studies, we show that VapC20 exists as a homodimer in solution. The structural analysis of VapC homologs further suggests that VapCs form homodimers. We demonstrate that VapC20 is an obligate homodimer, and its self-association is critical for its folding and activity. Surface plasmon resonance experiments suggest that VapC20 interacts with its cognate antitoxin VapB20 to form a stable complex with nanomolar affinity. A high association rate coupled with a very slow dissociation rate ensures minimal toxicity under normal growth conditions. AUC studies reveal that VapB20 also exists as a homodimer in solution and further associates with VapC20 dimers to form heterotetramers and heterooctamers in a concentration-dependent manner. The results presented here provide valuable insights into the assembly of VapBC family of toxins which is essential for their function and regulation.
Structural data are available in the PDB under the accession numbers 5WZF and 5WZ4.
VapBCs 是与毒力相关的蛋白，是原核生物中最丰富的 II 型毒素-抗毒素（TA）系统。在正常条件下，毒素和抗毒素相互作用形成异源八聚体复合物，该复合物与操纵子结合位点结合后，抑制自身表达。在应激条件下，VapB 抗毒素被细胞蛋白酶降解，释放出游离的 VapC 毒素，后者通过靶向蛋白翻译来抑制细胞生长。然而，异源八聚体复合物组装的中间步骤尚未得到解决。在这里，我们报告了来自结核分枝杆菌 II 型 TA 系统的 Sarcin-Ricin 环切割毒素 VapC20 的 1.75 Å 分辨率晶体结构。通过分析超速离心（AUC）研究，我们表明 VapC20 在溶液中以同源二聚体的形式存在。VapC 同源物的结构分析进一步表明 VapCs 形成同源二聚体。我们证明 VapC20 是一种必需的同源二聚体，其自身缔合对于其折叠和活性至关重要。表面等离子体共振实验表明，VapC20 与它的同源抗毒素 VapB20 相互作用，以纳米摩尔亲和力形成稳定的复合物。高缔合速率和非常缓慢的解离速率确保在正常生长条件下毒性最小。AUC 研究表明，VapB20 也以同源二聚体的形式存在于溶液中，并进一步与 VapC20 二聚体以浓度依赖的方式形成异源四聚体和异源八聚体。这里呈现的结果为 VapBC 家族毒素的组装提供了有价值的见解，这对于它们的功能和调节至关重要。
结构数据可在 PDB 中以 5WZF 和 5WZ4 的访问号获得。
