Wolfe David M, Zhang Yaoping, Roberts Gary P
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.
J Bacteriol. 2007 Oct;189(19):6861-9. doi: 10.1128/JB.00759-07. Epub 2007 Jul 20.
The nitrogen regulatory protein P(II) and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P(II) homolog, GlnJ, has higher affinity for an AmtB(1)-containing membrane than the other two P(II) homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB(1) complex in the presence of ATP and 2-KG, supporting a role for P(II) as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K(d) of 2-KG for GlnJ bound to AmtB(1) is 340 microM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 microM. This supports a model where multiple 2-KG and ATP molecules must bind a P(II) trimer to stimulate release of P(II) from AmtB(1), in contrast to the lower 2-KG requirement for productive uridylylation of P(II) by GlnD.
氮调节蛋白P(II)和氨气通道蛋白AmtB在大多数原核生物中均有发现。在多种生物体中都观察到了这两种蛋白质之间的相互作用，并且这种相互作用可能会调节这两种蛋白质的活性。目前对它们相互作用的调节机制仅了解一部分，我们发现，在红螺菌中，一种P(II)同源物GlnJ比另外两种P(II)同源物GlnB和GlnK对含有AmtB(1)的膜具有更高的亲和力。这种相互作用强烈倾向于GlnJ的非尿苷酸化形式，并且在没有ATP的情况下，高浓度的2-酮戊二酸（2-KG）或在有ATP的情况下低浓度的2-KG会破坏这种相互作用。在有ATP和2-KG存在时，ADP会抑制GlnJ-AmtB(1)复合物的不稳定，这支持了P(II)作为测量ATP与ADP比率的能量传感器的作用。在ATP饱和水平存在的情况下，与结合到AmtB(1)上的GlnJ结合的2-KG的估计解离常数（K(d)）为340微摩尔，这高于体外GlnJ尿苷酸化所需的浓度，约为5微摩尔。这支持了一个模型，即多个2-KG和ATP分子必须结合一个P(II)三聚体以刺激P(II)从AmtB(1)释放，这与GlnD对P(II)进行有效尿苷酸化所需的较低2-KG要求形成对比。
