Interfakultäres Institut für Mikrobiologie und Infektionsmedizin der Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
J Mol Biol. 2010 Jun 11;399(3):410-21. doi: 10.1016/j.jmb.2010.04.018. Epub 2010 Apr 24.
P(II) signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, P(II) binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, P(II) (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of P(II) binding to NAGK, we searched for P(II) variants with altered binding characteristics and found P(II) variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type P(II) protein. Analysis of interactions between these P(II) variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the P(II) I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the P(II) I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of P(II) proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus P(II) after having latched onto NAGK. Moreover, both P(II) I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of P(II)-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of P(II) initiates the bending of the extended T-loop of P(II), followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex.
P(II)信号转导蛋白在细菌、古菌和植物中高度保守，通过整合细胞碳、氮和能量状态的信号，在协调中心代谢方面发挥着关键作用。在集胞藻 PCC 7942 中，P(II)以协同的方式结合 ATP 和 2-氧戊二酸（2-OG），ATP 结合位点也接受 ADP。根据其效应分子结合状态，P(II)（来自这种蓝藻和其他需氧光养生物）复合物调节循环鸟氨酸途径的精氨酸控制酶，N-乙酰-L-谷氨酸激酶（NAGK），以控制精氨酸生物合成。为了更深入地了解 P(II)与 NAGK 结合的过程，我们寻找具有改变的结合特性的 P(II)变体，并发现 P(II)变体 I86N 和 I86T 能够与先前显示无法结合野生型 P(II)蛋白的 NAGK 变体（R233A）结合。这些 P(II)变体与野生型 NAGK 以及 NAGK R233A 变体之间相互作用的分析表明，P(II)I86N 变体是一种超活性 NAGK 结合物。为了揭示这种特性的结构基础，我们以原子分辨率解决了 P(II)I86N 变体的晶体结构。在大多数 P(II)蛋白受体相互作用中占主导地位的大 T 环呈现出紧密弯曲的构象，类似于与 NAGK 锁定后 S. elongatus P(II)的 T 环。此外，两种 P(II)I86 变体在 2-OG 结合中均显示出特定的缺陷，这表明残基 I86 在 2-OG 结合中起作用。我们提出了 P(II)-NAGK 复合物形成机制的两步模型：在起始步骤中，NAGK 的 R233 与 P(II)的 E85 之间的接触启动 P(II)的伸展 T 环的弯曲，随后是第二步，其中弯曲的 T 环深深插入 NAGK 的裂缝中形成紧密的复合物。
