MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
J Mol Biol. 2010 Aug 27;401(4):653-70. doi: 10.1016/j.jmb.2010.06.030. Epub 2010 Jun 19.
Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.
Lon ATP 依赖性蛋白酶是细菌细胞和真核细胞器蛋白质质量控制系统的关键组成部分。真细菌 Lon 蛋白酶包含一个 N 端结构域、一个 ATP 酶结构域和一个蛋白酶结构域，所有这些都在一条多肽链中。N 端结构域被认为参与底物识别，ATP 酶结构域参与底物展开和易位到蛋白酶腔，蛋白酶结构域参与将多肽水解成小肽片段。像其他 AAA+ATP 酶和自我分隔蛋白酶一样，Lon 作为寡聚体复合物发挥作用，尽管目前尚不清楚亚基化学计量。在这里，我们展示了来自枯草芽孢杆菌（BsLon）的 Lon 蛋白酶的截断版本的晶体结构，这些结构揭示了 Lon 复合物以前未知的结构特征。我们的分析超速离心和电子显微镜显示来自两种不同细菌物种，水生栖热菌和枯草芽孢杆菌的 Lon 蛋白酶的不同寡聚化。BsLon-AP 的结构显示出一个六聚体复合物，由一小部分 N 端结构域、ATP 酶和蛋白酶结构域组成。该结构显示了 Lon 的三个功能结构域的大致排列。它还揭示了 Lon 蛋白酶的结构与细菌蛋白酶体样蛋白酶 HslUV 的相似性。我们的第二个结构，BsLon-N，代表枯草芽孢杆菌 Lon N 端结构域的前 209 个氨基酸，由一个球状结构域组成，结构类似于大肠杆菌 Lon N 端结构域，以及另外四个螺旋束，这是预测的卷曲螺旋区域的一部分。BsLon-N 单体之间意外的二聚体相互作用表明 Lon 复合物可能通过相邻 N 端结构域之间的卷曲螺旋相互作用稳定。总的来说，BsLon-N 和 BsLon-AP 短了 36 个氨基酸，无法提供全长 Lon 蛋白酶的完整图片。
