Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700 019, India.
Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700 019, India.
Int J Biol Macromol. 2020 Jan 1;142:18-32. doi: 10.1016/j.ijbiomac.2019.11.217. Epub 2019 Nov 29.
Proper interaction between the divisome proteins FtsA and FtsZ is important for the bacterial cell division which is not well characterized till date. In this study, the objective was to understand the mechanism of FtsA-FtsZ interaction using full-length recombinant proteins. We cloned, over-expressed, purified and subsequently characterized FtsA of Vibrio cholerae (VcFtsA). We found that VcFtsA polymerization assembly was dependent on Ca ions, which is unique among FtsA proteins reported until now. VcFtsA also showed ATPase activity and its assembly was ATP dependent. Binding parameters of the interaction between the two full-length proteins, VcFtsA, and VcFtsZ determined by fluorescence spectrophotometry yielded a K value of around 38 μM. The K value of the interaction was 3 μM when VcFtsA was in ATP bound state. We found that VcFtsZ after interacting with VcFtsA causes a change of secondary structure in the later one leading to loss of its ability to hydrolyze ATP, subsequently halting the VcFtsA polymerization. On the other hand, a double-mutant of VcFtsA (VcFtsA-D242E,R300E), that does not bind to VcFtsZ, polymerized in the presence of VcFtsZ. Though FtsA proteins among different organisms show 70-80% homology in their sequences, assembly of VcFtsA showed a difference in its regulatory processes.
FtsA 和 FtsZ 蛋白之间的正确相互作用对于细菌分裂至关重要，但迄今为止其机制尚未得到很好的描述。本研究旨在使用全长重组蛋白来理解 FtsA-FtsZ 相互作用的机制。我们克隆、过表达、纯化并随后表征了霍乱弧菌（VcFtsA）的 FtsA。我们发现 VcFtsA 的聚合组装依赖于 Ca 离子，这在迄今为止报道的 FtsA 蛋白中是独特的。VcFtsA 还表现出 ATP 酶活性，其组装依赖于 ATP。荧光分光光度法测定的两个全长蛋白（VcFtsA 和 VcFtsZ）之间相互作用的结合参数得出 K 值约为 38 μM。当 VcFtsA 处于 ATP 结合状态时，相互作用的 K 值为 3 μM。我们发现，与 VcFtsA 相互作用后的 VcFtsZ 会导致后者二级结构发生变化，从而失去水解 ATP 的能力，随后停止 VcFtsA 的聚合。另一方面，不与 VcFtsZ 结合的 VcFtsA 双突变体（VcFtsA-D242E,R300E）在 VcFtsZ 存在的情况下聚合。尽管不同生物体的 FtsA 蛋白在其序列中具有 70-80%的同源性，但 VcFtsA 的组装在其调节过程中表现出差异。
