Sanofi Strasbourg R&D Center, Strasbourg, France.
Eli Lilly and Company, Cambridge, MA, USA.
Methods Mol Biol. 2022;2550:291-304. doi: 10.1007/978-1-0716-2593-4_31.
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule, which may act as scavenger of free radicals, and also to the binding of melatonin to its protein targets. For a long time, melatonin had been described as a ligand of a putative "receptor" present in the mammalian brain. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor," which led to the discovery of MT3 as the third melatonin binding site. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2). Among the different approaches used to validate that MT3 was indeed NQO2, the co-crystallization of NQO2 with melatonin was key in demonstrating the exact binding site and mode of melatonin to the enzyme and led to a clear understanding of the residues important for protein binding and inhibition. In this chapter, we described the details for the cloning, expression, and purification of the human enzyme NQO2. We also describe a detailed protocol for the crystallization of melatonin with this protein.
褪黑素（N-乙酰-5-甲氧基色胺）是一种神经激素，具有广泛的生物学效应。褪黑素介导的作用部分归因于该分子的抗氧化特性，其可以作为自由基清除剂，并且也归因于褪黑素与其蛋白靶标的结合。长期以来，褪黑素一直被描述为存在于哺乳动物大脑中的假定“受体”的配体。因此，进行了几项研究以阐明这种褪黑素“受体”的性质，这导致了 MT3 作为第三个褪黑素结合位点的发现。该结合位点被几个独立的小组证实，最终证明 MT3 是酶醌还原酶 2（NQO2）。在用于验证 MT3 确实是 NQO2 的不同方法中，NQO2 与褪黑素的共结晶在证明褪黑素与酶的精确结合位点和方式方面起着关键作用，并导致对蛋白质结合和抑制的重要残基的清晰理解。在本章中，我们描述了人酶 NQO2 的克隆、表达和纯化的详细过程。我们还描述了使用该蛋白结晶褪黑素的详细方案。
