MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein
MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein
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DOI：
10.1128/jb.181.15.4628-4638.1999
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发表时间：
1999-08-01
影响因子：
3.2
通讯作者：
Kustu, S
中科院分区：
文献类型：
--
作者：
Rombel, I;Peters-Wendisch, P;Kustu, S
当磷酸化时，鼠伤寒沙门氏菌的氮调节蛋白C（NtrC）的二聚体形式形成较大的寡聚体，所述寡聚体可以水解ATP，从而通过RNA聚合酶的σ（54）全酶形式激活转录。镁-核苷三磷酸结合的研究，使用过滤器结合试验表明，磷酸化是不需要的核苷酸结合，但可能控制核苷酸水解本身。通过等温滴定量热法进行的结合研究表明，在25 ℃下，未磷酸化的NtrC对MgATP γ S的表观Kd为100 μ M，通过滤膜结合进行的研究表明，在37 ℃下，半最大结合所需的MgATP浓度为130 μ M。过滤器结合的研究与突变形式的NtrC缺陷ATP水解牵连两个地区的中央结构域直接在核苷酸结合和三个额外的区域水解。所有五个在sigma（54）-全酶的激活剂中是高度保守的。参与结合的区域是步行者A基序和残基G355至R358周围的区域，其可与核苷酸碱基相互作用。参与核苷酸水解的区域是残基S207和E208，它们被认为位于与p21（ras）和其他嘌呤核苷酸结合蛋白的开关I效应区类似的区域;残基R294，其可能是催化残基;和残基D239，其是推定的步行者B基序中的保守天冬氨酸。D239似乎在结合核苷酸水解所必需的二价阳离子中起作用。Mn ~（2+）结合的电子顺磁共振分析表明，在不存在核苷酸的情况下，NtrC的中心结构域不强烈地结合二价阳离子。
When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of Salmonella typhimurium forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the sigma(54)-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent K-d of unphosphorylated NtrC for MgATP gamma S is 100 mu M at 25 degrees C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 mu M at 37 degrees C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of sigma(54)-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21(ras) and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn2+ binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.