The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis
The Molecular Basis of TnrA Control by Glutamine Synthetase in Bacillus subtilis
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DOI：
10.1074/jbc.m115.680991
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发表时间：
2016-02-12
影响因子：
4.8
通讯作者：
Forchhammer, Karl
中科院分区：
文献类型：
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作者：
Hauf, Ksenia;Kayumov, Airat;Forchhammer, Karl
TnrA是枯草芽孢杆菌氮同化的主要调控因子。本研究的重点是谷氨酰胺合成酶（GS）如何抑制TnrA功能，以响应关键代谢产物ATP、AMP、谷氨酰胺和谷氨酸的机制。我们提出了一个两种相互排斥的GS构象控制与TnrA相互作用的模型。在atp结合状态（a态）下，GS具有催化活性，但不能与TnrA相互作用。磷酸化的l-蛋氨酸亚砜胺（MSX）稳定了这种构象，将酶固定在过渡状态。当被谷氨酰胺（或其类似物MSX）占据时，GS处于对TnrA （q态）具有高亲和力的构象中。A态和q态是相互排斥的，ATP和谷氨酰胺以竞争的方式结合到GS上。在谷氨酰胺浓度升高时，ATP不再能够结合GS并使其进入a态。AMP有效地与ATP竞争并阻止a态的形成，从而有利于GS-TnrA相互作用。表面等离子体共振分析表明，与正调控启动子片段结合的TnrA以q态结合GS，而与负调控启动子片段迅速分离。这些数据表明，GS通过屏蔽处于dna结合状态的转录因子来控制TnrA活性。根据尺寸不相容和多角度光散射分析，十二聚体GS可以结合三种TnrA二聚体。GS的高度相互依赖的配体结合特性揭示了这种酶作为细胞氮和能量状态的复杂传感器来控制dna结合的TnrA的活性。
TnrA is a master regulator of nitrogen assimilation in Bacillus subtilis. This study focuses on the mechanism of how glutamine synthetase (GS) inhibits TnrA function in response to key metabolites ATP, AMP, glutamine, and glutamate. We suggest a model of two mutually exclusive GS conformations governing the interaction with TnrA. In the ATP-bound state (A-state), GS is catalytically active but unable to interact with TnrA. This conformation was stabilized by phosphorylated l-methionine sulfoximine (MSX), fixing the enzyme in the transition state. When occupied by glutamine (or its analogue MSX), GS resides in a conformation that has high affinity for TnrA (Q-state). The A- and Q-state are mutually exclusive, and in agreement, ATP and glutamine bind to GS in a competitive manner. At elevated concentrations of glutamine, ATP is no longer able to bind GS and to bring it into the A-state. AMP efficiently competes with ATP and prevents formation of the A-state, thereby favoring GS-TnrA interaction. Surface plasmon resonance analysis shows that TnrA bound to a positively regulated promoter fragment binds GS in the Q-state, whereas it rapidly dissociates from a negatively regulated promoter fragment. These data imply that GS controls TnrA activity at positively controlled promoters by shielding the transcription factor in the DNA-bound state. According to size exclusion and multiangle light scattering analysis, the dodecameric GS can bind three TnrA dimers. The highly interdependent ligand binding properties of GS reveal this enzyme as a sophisticated sensor of the nitrogen and energy state of the cell to control the activity of DNA-bound TnrA.