Department of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University.
J Gen Appl Microbiol. 2021 Feb 26;66(6):297-306. doi: 10.2323/jgam.2019.12.003. Epub 2020 May 19.
An Escherichia coli ATP-dependent two-component protease, ClpYQ(HslUV), targets the SulA molecule, an SOS induced protein. ClpY recognizes, unfolds and translocates the substrates into the proteolytic site of ClpQ for degradation. ClpY is divided into three domains N, I and C. The N domain is an ATPase; the C domain allows for oligomerization, while the I domain coordinates substrate binding. In the ClpYQ complex, two layer pore sites, pore I and II, are in the center of its hexameric rings. However, the actual roles of two outer-loop (130159 aa, L1 and 175209 aa, L2) of the ClpY-I domain for the degradation of SulA are unclear. In this study, with ATP, the MBP-SulA molecule was bound to ClpY oligomer(s). ClpYΔL1 (ClpY deleted of loop 1) oligomers revealed an excessive SulA-binding activity. With ClpQ, it showed increased proteolytic activity for SulA degradation. Yet, ClpYΔL2 formed fewer oligomers that retained less proteolytic activity, but still had increased SulA-binding activity. In contrast, ClpYΔpore I had a lower SulA-binding activity. ClpYΔ pore I ΔL2 showed the lowest SulA-binding activity. In addition, ClpY (Q198L, Q200L), with a double point mutation in loop 2, formed stable oligomers. It also had a subtle increase in SulA-binding activity, but displayed less proteolytic activity. As a result, loop 2 has an effect on ClpY oligomerization, substrate binding and delivery. Loop 1 has a role as a gate, to prevent excessive substrate binding. Thus, accordingly, ClpY permits the formation of SulA-ClpY, with ATP(s), and this complex then docks through ClpQ for ultimate proteolytic degradation.
大肠杆菌依赖于 ATP 的双组分蛋白酶 ClpYQ（HslUV）靶向 SulA 分子，这是一种 SOS 诱导的蛋白质。ClpY 识别、展开并将底物转运到 ClpQ 的蛋白酶切位点进行降解。ClpY 分为三个结构域：N、I 和 C。N 结构域是一个 ATP 酶；C 结构域允许寡聚化，而 I 结构域协调底物结合。在 ClpYQ 复合物中，两个层状孔位点，孔 I 和孔 II，位于其六聚体环的中心。然而，ClpY-I 结构域两个外环（130159aa，L1 和 175209aa，L2）对于 SulA 降解的实际作用尚不清楚。在这项研究中，在 ATP 存在的情况下，MBP-SulA 分子与 ClpY 寡聚体结合。ClpYΔL1（ClpY 缺失环 1）寡聚体显示出过度的 SulA 结合活性。与 ClpQ 一起，它显示出 SulA 降解的增强的蛋白水解活性。然而，ClpYΔL2 形成的寡聚体较少，保留的蛋白水解活性较低，但仍具有增强的 SulA 结合活性。相比之下，ClpYΔpore I 具有较低的 SulA 结合活性。ClpYΔpore IΔL2 显示出最低的 SulA 结合活性。此外，ClpY（Q198L，Q200L）在环 2 中有一个双点突变，形成稳定的寡聚体。它还具有轻微增加的 SulA 结合活性，但显示出较低的蛋白水解活性。因此，环 2 对 ClpY 寡聚化、底物结合和递呈有影响。环 1 充当门，以防止过度的底物结合。因此，ClpY 允许 SulA-ClpY 的形成，与 ATP（s）结合，然后通过 ClpQ 对接，最终进行蛋白水解降解。
