Radchenko Martha V, Thornton Jeremy, Merrick Mike
Department of Molecular Microbiology, John Innes Centre Norwich, UK.
Front Microbiol. 2014 Dec 23;5:731. doi: 10.3389/fmicb.2014.00731. eCollection 2014.
PII proteins are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and their regulatory effect is achieved by direct interaction with their target. Many, but by no means all, PII proteins are subject to post-translational modification of a residue within the T-loop of the protein. The protein's modification state is influenced by the cellular nitrogen status and in the past this has been considered to regulate PII activity by controlling interaction with target proteins. However, the fundamental ability of PII proteins to respond to the cellular nitrogen status has been shown to be dependent on binding of key effector molecules, ATP, ADP, and 2-oxoglutarate which brings into question the precise role of post-translational modification. In this study we have used the Escherichia coli PII protein GlnK to examine the influence of post-translational modification (uridylylation) on the interaction between GlnK and its cognate target the ammonia channel protein AmtB. We have compared the interaction with AmtB of wild-type GlnK and a variant protein, GlnKTyr51Ala, that cannot be uridylylated. This analysis was carried out both in vivo and in vitro and showed that association and dissociation of the GlnK-AmtB complex is not dependent on the uridylylation state of GlnK. However, our in vivo studies show that post-translational modification of GlnK does influence the dynamics of its interaction with AmtB.
PII蛋白是细菌和古细菌中氮代谢调控的关键参与者，在植物的质体中也有发现。PII蛋白控制着多种酶、转录因子和膜转运蛋白的活性，其调节作用是通过与靶标直接相互作用实现的。许多（但绝非全部）PII蛋白会在蛋白质T环内的一个残基上发生翻译后修饰。蛋白质的修饰状态受细胞氮状态的影响，过去人们认为这是通过控制与靶蛋白的相互作用来调节PII活性。然而，已表明PII蛋白响应细胞氮状态的基本能力取决于关键效应分子ATP、ADP和2-氧代戊二酸的结合，这使得翻译后修饰的确切作用受到质疑。在本研究中，我们使用大肠杆菌PII蛋白GlnK来研究翻译后修饰（尿苷酸化）对GlnK与其同源靶标氨通道蛋白AmtB之间相互作用的影响。我们比较了野生型GlnK和不能被尿苷酸化的变体蛋白GlnKTyr51Ala与AmtB的相互作用。这项分析在体内和体外均进行，结果表明GlnK-AmtB复合物的结合和解离不依赖于GlnK的尿苷酸化状态。然而，我们的体内研究表明，GlnK的翻译后修饰确实会影响其与AmtB相互作用的动力学。
