Centro de Investigaciones Biológicas Margarita Salas CSIC, Madrid, Spain.
Rocasolano Institute for Physical Chemistry CSIC, Madrid, Spain.
FEBS J. 2020 Sep;287(18):4048-4067. doi: 10.1111/febs.15235. Epub 2020 Feb 24.
The essential bacterial division protein FtsZ uses GTP binding and hydrolysis to assemble into dynamic filaments that treadmill around the Z-ring, guiding septal wall synthesis and cell division. FtsZ is a structural homolog of tubulin and a target for discovering new antibiotics. Here, using FtsZ from the pathogen S. aureus (SaFtsZ), we reveal that, prior to assembly, FtsZ monomers require nucleotide binding for folding; this is possibly relevant to other mesophilic FtsZs. Apo-SaFtsZ is essentially unfolded, as assessed by nuclear magnetic resonance and circular dichroism. Binding of GTP (≥ 1 mm) dramatically shifts the equilibrium toward the active folded protein. Supportingly, SaFtsZ refolded with GDP crystallizes in a native structure. Apo-SaFtsZ also folds with 3.4 m glycerol, enabling high-affinity GTP binding (K 20 nm determined by isothermal titration calorimetry) similar to thermophilic stable FtsZ. Other stabilizing agents that enhance nucleotide binding include ethylene glycol, trimethylamine N-oxide, and several bacterial osmolytes. High salt stabilizes SaFtsZ without bound nucleotide in an inactive twisted conformation. We identified a cavity behind the SaFtsZ-GDP nucleotide-binding pocket that harbors different small compounds, which is available for extended nucleotide-replacing inhibitors. Furthermore, we devised a competition assay to detect any inhibitors that overlap the nucleotide site of SaFtsZ, or Escherichia coli FtsZ, employing osmolyte-stabilized apo-FtsZs and the specific fluorescence anisotropy change in mant-GTP upon dissociation from the protein. This robust assay provides a basis to screening for high-affinity GTP-replacing ligands, which combined with structural studies and phenotypic profiling should facilitate development of a next generation of FtsZ-targeting antibacterial inhibitors.
必需的细菌分裂蛋白 FtsZ 使用 GTP 结合和水解作用组装成动态丝，在 Z 环周围进行“履带式”运动，指导隔膜壁的合成和细胞分裂。FtsZ 是微管蛋白的结构同源物，也是发现新抗生素的靶点。在这里，我们使用病原体金黄色葡萄球菌（SaFtsZ）的 FtsZ 揭示了，在组装之前，FtsZ 单体需要核苷酸结合才能折叠；这可能与其他嗜温性 FtsZ 有关。通过核磁共振和圆二色性评估，无核苷酸结合的 apo-SaFtsZ 基本上是未折叠的。GTP（≥1mm）的结合极大地将平衡转向活性折叠蛋白。支持这一观点的是，与 GDP 复性的 SaFtsZ 结晶为天然结构。apo-SaFtsZ 也可以与 3.4m 甘油折叠，从而能够进行高亲和力的 GTP 结合（通过等温滴定量热法测定的 K 20nm），类似于耐热稳定的 FtsZ。其他增强核苷酸结合的稳定剂包括乙二醇、三甲胺 N-氧化物和几种细菌渗透剂。高盐在无结合核苷酸的情况下稳定处于非活性扭曲构象的 SaFtsZ。我们在 SaFtsZ-GDP 核苷酸结合口袋的后面发现了一个空腔，其中包含不同的小分子，这些小分子可用于扩展核苷酸替代抑制剂。此外，我们设计了一种竞争测定法来检测任何重叠 SaFtsZ 或大肠杆菌 FtsZ 核苷酸结合位点的抑制剂，方法是使用渗透剂稳定的 apo-FtsZ 和从蛋白上解离时 mant-GTP 的特异性荧光各向异性变化。这种强大的测定法为筛选高亲和力 GTP 替代配体提供了基础，与结构研究和表型分析相结合，应有助于开发下一代靶向 FtsZ 的抗菌抑制剂。
