Farez-Vidal M E, Wilson T J, Davidson B E, Howlett G J, Austin S, Dixon R A
Nitrogen Fixation Laboratory, John Innes Centre, Norwich, UK.
Mol Microbiol. 1996 Dec;22(5):779-88. doi: 10.1046/j.1365-2958.1996.01530.x.
The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of sigma N-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATP gamma S concentration suggested an apparent dissociation constant (Kd) for ATP gamma S of 150 microM. In the presence of DNA, both the central and C-terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change. NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NTRC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NTRC displayed a monomer-dimer equilibrium with a Kd of 6 microM. In the presence of ATP gamma S the equilibrium was shifted towards the dimer form (Kd = 0.8 microM). A similar dissociation constant for the monomer-dimer interaction was observed with NTRC S160F in the absence of ATP gamma S (Kd = 0.5 microM). The addition of ATP gamma S induced a significant association of NTRC S160F to higher-order states with a dimer-octamer model producing a slightly, but not significantly better fit to the data than a dimer-hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.
肺炎克雷伯菌氮调节蛋白NTRC是一种响应调节因子，可响应氮限制激活转录，并且是σN依赖性增强子结合蛋白家族的成员。通过有限的胰蛋白酶消化，检测到NTRC的两个结构域，并且还观察到蛋白质内响应配体结合的构象变化。在没有配体的情况下，主要消化产物分别是对应于中央加C末端结构域、中央结构域和N末端结构域的42、36和12.5 kDa条带。在嘌呤而非嘧啶核苷酸结合后，36 kDa条带对进一步的蛋白水解不敏感，表明中央结构域发生了构象变化。对这种不敏感性对ATPγS浓度的依赖性分析表明，ATPγS的表观解离常数（Kd）为150μM。在存在DNA的情况下，NTRC的中央和C末端结构域对蛋白水解切割均不敏感，表明发生了进一步的构象变化。NTRC S160F是NTRC的一种突变形式，在没有磷酸化的情况下具有活性，比野生型蛋白对蛋白水解更稳定。这种突变蛋白显然被锁定在类似于野生型NTRC与DNA结合形式的构象中。使用沉降平衡分析研究了配体在自缔合中的作用。在没有配体的情况下，野生型NTRC显示出单体-二聚体平衡，Kd为6μM。在存在ATPγS的情况下，平衡向二聚体形式移动（Kd = 0.8μM）。在没有ATPγS的情况下，NTRC S160F的单体-二聚体相互作用也观察到类似的解离常数（Kd = 0.5μM）。添加ATPγS诱导NTRC S160F显著缔合为高阶状态，二聚体-八聚体模型对数据的拟合略好于二聚体-六聚体模型，但差异不显著。我们提出配体介导的自缔合为这类转录调节蛋白的激活提供了一种共同机制。
