Skretas Georgios, Meligova Aggeliki K, Villalonga-Barber Carolina, Mitsiou Dimitra J, Alexis Michael N, Micha-Screttas Maria, Steele Barry R, Screttas Constantinos G, Wood David W
Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544, USA.
J Am Chem Soc. 2007 Jul 11;129(27):8443-57. doi: 10.1021/ja067754j. Epub 2007 Jun 15.
Engineered protein-based sensors of ligand binding have emerged as attractive tools for the discovery of therapeutic compounds through simple screening systems. We have previously shown that engineered chimeric enzymes, which combine the ligand-binding domains of nuclear hormone receptors with a highly sensitive thymidylate synthase reporter, yield simple sensors that report the presence of hormone-like compounds through changes in bacterial growth. This work describes an optimized estrogen sensor in Escherichia coli with extraordinary reliability in identifying diverse estrogenic compounds and in differentiating between their agonistic/antagonistic pharmacological effects. The ability of this system to assist the discovery of new estrogen-mimicking compounds was validated by screening a small compound library, which led to the identification of two structurally novel estrogen receptor modulators and the accurate prediction of their agonistic/antagonistic biocharacter in human cells. Strong evidence is presented here that the ability of our sensor to detect ligand binding and recognize pharmacologically critical properties arises from allosteric communication between the artificially combined protein domains, where different ligand-induced conformational changes in the receptor are transmitted to the catalytic domain and translated to distinct levels of enzymic efficiency. To the best of our knowledge, this is one of the first examples of an engineered enzyme with the ability to sense multiple receptor conformations and to be either activated or inactivated depending on the nature of the bound effector molecule. Because the proposed mechanism of ligand dependence is not specific to nuclear hormone receptors, we anticipate that our protein engineering strategy will be applicable to the construction of simple sensors for different classes of (therapeutic) binding proteins.
基于工程蛋白的配体结合传感器已成为通过简单筛选系统发现治疗性化合物的有吸引力的工具。我们之前已经表明，将核激素受体的配体结合结构域与高灵敏度的胸苷酸合酶报告基因相结合的工程嵌合酶，能产生简单的传感器，通过细菌生长变化报告类激素化合物的存在。这项工作描述了一种在大肠杆菌中优化的雌激素传感器，它在识别多种雌激素化合物以及区分它们的激动/拮抗药理作用方面具有非凡的可靠性。通过筛选一个小分子化合物库，验证了该系统协助发现新型雌激素模拟化合物的能力，这导致鉴定出两种结构新颖的雌激素受体调节剂，并准确预测了它们在人细胞中的激动/拮抗生物特性。这里提供了强有力的证据表明，我们的传感器检测配体结合并识别药理关键特性的能力源于人工组合的蛋白质结构域之间的变构通讯，其中受体中不同的配体诱导构象变化被传递到催化结构域并转化为不同水平的酶效率。据我们所知，这是具有感知多种受体构象并根据结合的效应分子性质被激活或失活能力的工程酶的首批实例之一。由于所提出的配体依赖性机制并非核激素受体所特有，我们预计我们的蛋白质工程策略将适用于构建针对不同类别的（治疗性）结合蛋白的简单传感器。
