Ikegami T, Okada T, Ohki I, Hirayama J, Mizuno T, Shirakawa M
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Biochemistry. 2001 Jan 16;40(2):375-86. doi: 10.1021/bi001619g.
An Escherichia coli sensor kinase, ArcB, transfers a phosphoryl group to a partner response regulator in response to anaerobic conditions. Multidimensional NMR techniques were applied to determine the solution structure of the histidine-containing phosphotransfer signaling domain of ArcB (HPt(ArcB)), which has a phosphorylation site, His717. The backbone dynamics were also investigated by analyses of the (15)N relaxation data and amide hydrogen exchange rates. Furthermore, the protonation states of the histidine imidazole rings were characterized by means of (1)H and (15)N chemical shifts at various pHs. The determined solution structure of HPt(ArcB) contains five helices and forms a four-helix bundle motif like other HPt domains. The obtained order parameters, S (2), [(1)H]-(15)N heteronuclear NOE values, and chemical exchange parameters, R(ex), showed that the alpha-helical regions of HPt(ArcB) are rigid on both picosecond to nanosecond and microsecond to millisecond time scales. On the other hand, helix D, which contains His717, exhibited low protection factors of less than 4000, indicating the presence of fluctuations on a slower time scale in helix D. These results suggest that HPt(ArcB) may undergo a small conformational change in helix D upon phosphorylation. It was also shown that the imidazole ring of His717 has a pK(a) value of 6.76, which is similar to that of a solvent-exposed histidine imidazole ring, and that a pair of deprotonated neutral tautomers are rapidly exchanged with each other. This is consistent with the solution structure of HPt(ArcB), in which the imidazole ring of His717 is exposed to the solvent.
一种大肠杆菌传感激酶ArcB，在厌氧条件下会将磷酸基团转移至伙伴应答调节蛋白。采用多维核磁共振技术来确定ArcB含组氨酸的磷酸转移信号结构域（HPt(ArcB)）的溶液结构，该结构域有一个磷酸化位点His717。还通过分析¹⁵N弛豫数据和酰胺氢交换速率来研究其主链动力学。此外，借助不同pH值下的¹H和¹⁵N化学位移对组氨酸咪唑环的质子化状态进行了表征。所确定的HPt(ArcB)溶液结构包含5个螺旋，并且像其他HPt结构域一样形成了四螺旋束基序。获得的序参数S²、¹H-¹⁵N异核NOE值以及化学交换参数Rex表明，HPt(ArcB)的α螺旋区域在皮秒到纳秒以及微秒到毫秒时间尺度上都是刚性的。另一方面，包含His717的螺旋D显示出小于4000的低保护因子，表明螺旋D中存在较慢时间尺度的波动。这些结果表明，HPt(ArcB)在磷酸化时可能在螺旋D中发生小的构象变化。还表明His717的咪唑环的pKa值为6.76，这与溶剂暴露的组氨酸咪唑环的pKa值相似，并且一对去质子化的中性互变异构体彼此快速交换。这与HPt(ArcB)的溶液结构一致，其中His717的咪唑环暴露于溶剂中。
