Zhu J, Winans S C
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1507-12. doi: 10.1073/pnas.98.4.1507.
Complexes between the quorum-sensing regulator TraR and its inducing ligand autoinducer (AAI) are soluble in Escherichia coli, whereas apo-TraR is almost completely insoluble. Here we show that the lack of soluble TraR is due in large part to rapid proteolysis, inasmuch as apo-TraR accumulated to high levels in an E. coli strain deficient in Clp and Lon proteases. In pulse labeling experiments, AAI protected TraR against proteolysis only when it was added before the radiolabel. This observation indicates that TraR proteins can productively bind AAI only during their own synthesis on polysomes, whereas fully synthesized apo-TraR proteins are not functional AAI receptors. Purified apo-TraR was rapidly degraded by trypsin to oligopeptides, whereas TraR-AAI complexes were more resistant to trypsin and were cleaved at discrete interdomain linkers, indicating that TraR requires AAI to attain its mature tertiary structure. TraR-AAI complexes eluted from a gel filtration column as dimers and bound DNA as dimers. In contrast, apo-TraR was monomeric, and incubation with AAI under a variety of conditions did not cause dimerization. We conclude that AAI is critical for the folding of nascent TraR protein into its mature tertiary structure and that full-length apo-TraR cannot productively bind AAI and is consequently targeted for rapid proteolysis.
群体感应调节因子TraR与其诱导配体自诱导物（AAI）之间的复合物在大肠杆菌中是可溶的，而无配体的TraR几乎完全不溶。在这里我们表明，可溶性TraR的缺乏在很大程度上是由于快速的蛋白水解作用，因为在缺乏Clp和Lon蛋白酶的大肠杆菌菌株中，无配体的TraR能积累到高水平。在脉冲标记实验中，只有当AAI在放射性标记之前添加时，它才能保护TraR不被蛋白水解。这一观察结果表明，TraR蛋白只有在其自身在多核糖体上合成期间才能有效地结合AAI，而完全合成的无配体TraR蛋白不是功能性的AAI受体。纯化的无配体TraR被胰蛋白酶迅速降解为寡肽，而TraR-AAI复合物对胰蛋白酶更具抗性，并在离散的结构域间连接部位被切割，这表明TraR需要AAI来获得其成熟的三级结构。从凝胶过滤柱上洗脱下来的TraR-AAI复合物以二聚体形式存在，并以二聚体形式结合DNA。相比之下，无配体的TraR是单体，在各种条件下与AAI孵育都不会导致二聚化。我们得出结论，AAI对于新生的TraR蛋白折叠成其成熟的三级结构至关重要，全长的无配体TraR不能有效地结合AAI，因此会被快速蛋白水解作用靶向降解。
