Sensation and signaling of alpha-ketoglutarate and adenylylate energy charge by the Escherichia coli PII signal transduction protein require cooperation of the three ligand-binding sites within the PII trimer.
Sensation and signaling of alpha-ketoglutarate and adenylylate energy charge by the Escherichia coli PII signal transduction protein require cooperation of the three ligand-binding sites within the PII trimer.
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DOI：
10.1021/bi9011594
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发表时间：
2009-12-08
期刊：
影响因子：
2.9
通讯作者：
Ninfa, Alexander J.
中科院分区：
文献类型：
--
作者：
Jiang, Peng;Ninfa, Alexander J.
PII 蛋白是 α-酮戊二酸和腺苷酸能量电荷的传感器，调节信号转导蛋白、代谢酶和参与氮同化的渗透酶。在这里，纯化的大肠杆菌 PII 及其两种受体 ATase 和 NRII 用于研究 PII 的感觉机制。我们从野生型和突变亚基组装了 PII 的异三聚体形式，这使我们能够评估 PII 三聚体中 α-酮戊二酸和腺苷酸核苷酸的三个结合位点的作用。这些异源三聚体 PII 蛋白发出的 α-酮戊二酸和腺苷酸能量电荷信号需要这些效应子的多个结合位点，并且不同亚基上的配体结合位点可能会影响单个亚基与受体相互作用的功能，这意味着 PII 亚基之间存在通信。 PII 的野生型和异源三聚体形式也用于检查 α-酮戊二酸和 ADP 对 PII 激活 ATase 的腺苷酸转移酶 (AT) 活性的影响。先前的研究表明，当 ATP 是唯一的腺苷酸核苷酸时，α-酮戊二酸控制 PII 激活的程度，但不会改变 PII 激活常数 (Kact)。我们发现 ADP 影响 PII Kact 和 PII 激活的程度。当 ATP 存在时，ADP 显着降低野生型 PII 的 Kact，并且这种作用被 α-酮戊二酸拮抗。因此，当 ATP 存在时，ADP 和 α-酮戊二酸之间的拮抗作用使这些效应子能够影响 PII Kact 以激活 ATase。对 PII 异三聚体形式的研究表明，ADP 改善 PII 与 ATase 结合的能力的主要部分需要多个核苷酸结合位点和亚基间通讯。我们还使用非变性凝胶电泳来研究 ADP 和 α-酮戊二酸对 PII 与 ATase 和 NRII 结合的影响。这些研究表明，ATase 和 NRII 与 PII 相互作用的要求不同，并且在适当的条件下，α-酮戊二酸和 ADP 之间的拮抗作用使这些效应子能够影响 PII 与受体的结合。
PII proteins are sensors of α-ketoglutarate and adenylylate energy charge that regulate signal transduction proteins, metabolic enzymes, and permeases involved in nitrogen assimilation. Here, purified Escherichia coli PII and two of its receptors, ATase and NRII, were used to study the mechanisms of sensation by PII. We assembled heterotrimeric forms of PII from wild-type and mutant subunits, which allowed us to assess the role of the three binding sites for α-ketoglutarate and adenylylate nucleotide in the PII trimer. Signaling of α-ketoglutarate and adenylylate energy charge by these heterotrimeric PII proteins required multiple binding sites for these effectors, and the ligand-binding sites on different subunits could influence the function of a single subunit interacting with a receptor, implying communication between PII subunits. Wild-type and heterotrimeric forms of PII were also used to examine the effects of α-ketoglutarate and ADP on PII activation of the adenylyltransferase (AT) activity of ATase. Previous work showed that when ATP was the sole adenylylate nucleotide, α-ketoglutarate controlled the extent of PII activation but did not alter the PII activation constant (Kact). We show that ADP affected both the PII Kact and the extent of activation by PII. When ATP was present, ADP dramatically reduced the Kact for wild-type PII, and this effect was antagonized by α-ketoglutarate. Consequently, when ATP was present, the antagonism between ADP and α-ketoglutarate allowed each of these effectors to influence the PII Kact for activation of ATase. A study of heterotrimeric forms of PII suggested that the major part of the ability of ADP to improve the binding of PII to ATase required multiple nucleotide binding sites and intersubunit communication. We also used nondenaturing gel electrophoresis to investigate the effect of ADP and α-ketoglutarate on the binding of PII to ATase and NRII. These studies showed that ATase and NRII differ in their requirements for interaction with PII, and that under the appropriate conditions, the antagonism between α-ketoglutarate and ADP allowed each of these effectors to influence the binding of PII to receptors.
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影响因子：
2.9
作者：
Jiang, Peng;Ninfa, Alexander J.
通讯作者：
Ninfa, Alexander J.
影响因子：
4.8
作者：
KAMBEROV, ES;ATKINSON, MR;NINFA, AJ
通讯作者：
NINFA, AJ
影响因子：
3
作者：
Sakai, H;Wang, HF;Yokoyama, S
通讯作者：
Yokoyama, S
DOI：
10.1046/j.1432-1327.2001.02074.x
发表时间：
2001-04-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Xu, YB;Carr, PD;Ollis, DL
通讯作者：
Ollis, DL
影响因子：
3.2
作者：
Jiang, P;Ninfa, AJ
通讯作者：
Ninfa, AJ