探测应答调控蛋白PhoBNF20D自由态中存在的Pre-Active构象

doi:10.11938/cjmr20182639

波谱学杂志 ›› 2019, Vol. 36 ›› Issue (2): 164-171.doi: 10.11938/cjmr20182639

探测应答调控蛋白PhoB_N^F20D自由态中存在的Pre-Active构象

寇新慧^1,2, 刘乙祥¹, 刘兴弘^1,2, 李从刚¹, 刘买利¹, 姜凌¹

1. 中国科学院生物磁共振重点实验室, 波谱与原子分子物理国家重点实验室, 武汉磁共振中心(中国科学院武汉物理与数学研究所), 湖北武汉 430071;
2. 中国科学院大学, 北京 100049

收稿日期:2018-04-20 发布日期:2018-05-18
通讯作者: 刘乙祥 Tel:027-87198493, E-mail:yixiangliu@wipm.ac.cn;姜凌 Tel:027-87198493, E-mail:lingjiang@wipm.ac.cn E-mail:yixiangliu@wipm.ac.cn;lingjiang@wipm.ac.cn
基金资助:
the National Key R&D Program of China (2017YFA0505400); the National Science Foundation of China (21573280, 21603268).

Visualizing the Pre-Active Conformation of Response Regulator PhoB_N^F20D in Its apo State

KOU Xin-hui^1,2, LIU Yi-xiang¹, LIU Xing-hong^1,2, LI Cong-gang¹, LIU Mai-li¹, JIANG Ling¹

1. CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan(Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences), Wuhan 430071, China;
2. University of Chinese Academy of Science, Beijing 100049, China

Received:2018-04-20 Published:2018-05-18
Supported by:
the National Key R&D Program of China (2017YFA0505400); the National Science Foundation of China (21573280, 21603268).

摘要/Abstract

摘要： PhoB是PhoR/PhoB双组分信号转导系统（TCS）中的应答调控蛋白，来自大肠杆菌，可以参与环境中自由Pi的调控应答.PhoB拥有保守的磷酸化位点D53，当Mg²⁺存在时，会变成活性构象，但是其中的机理尚不清楚.目前已经有报道指出，自由态的应答调控蛋白存在少量的活性构象，而且它们和蛋白的激活过程相关，但是这种活性构象一般不稳定，很难观测到.本文以PhoB_N^F20D作为模型，¹H-¹⁵N HSQC和¹⁹F NMR谱图显示，自由态PhoB_N^F20D在溶液中存在pre-active构象，BeF₃^-可以稳定这种活性构象，而且Mg²⁺可以促进非活性构象到活性构象之间的转化，在BeF₃^-存在的情况下可以使PhoB_N^F20D形成完全的活性构象.同时我们利用Carr-Purcell-Meiboom-Gill（CPMG）弛豫弥散实验证明了PhoB_N^F20D存在两态交换.

关键词: BeF₃^-, 活性构象, 核磁共振(NMR), 应答调控蛋白, 双组分信号转导系统(TCS)

Abstract: PhoB is a response regulator of the PhoR/PhoB two-component system (TCS) in Escherichia Coli, which is involved in environmental phosphate regulation. PhoB activation is known to be mediated by Mg²⁺ coordination and phosphorylation of the conserved D53 residue, but the exact mechanism remains to be elucidated. It has been proposed that minor active conformations of the response regulator may preexist in its apo state and be related to the activation. However, such minor states are instable and difficult to observe. In this work, ¹H-¹⁵N HSQC and ¹⁹F NMR experiments were performed on a monomeric mutant PhoB_N^F20D. It was observed that the apo PhoB_N^F20D had a pre-active conformation, which could be partially stabilized by the phosphoryl group analog BeF₃^-without the presence of Mg²⁺. Addition of Mg²⁺ shifted the equilibrium towards the active form. Furthermore, the exchange between the inactive and active conformations were confirmed by the CPMG (Carr-Purcell-Meiboom-Gill) relaxation dispersion experiments. It is concluded that the apo PhoB_N^F20D have both inactive and active conformations, and the pre-active conformation can be stabilized by BeF₃^-.

Key words: BeF₃^-, active conformation, nuclear magnetic resonance (NMR), response regulator, two-component system (TCS)

中图分类号:

O482.53

寇新慧, 刘乙祥, 刘兴弘, 李从刚, 刘买利, 姜凌. 探测应答调控蛋白PhoB_N^F20D自由态中存在的Pre-Active构象[J]. 波谱学杂志, 2019, 36(2): 164-171.

KOU Xin-hui, LIU Yi-xiang, LIU Xing-hong, LI Cong-gang, LIU Mai-li, JIANG Ling. Visualizing the Pre-Active Conformation of Response Regulator PhoB_N^F20D in Its apo State[J]. Chinese Journal of Magnetic Resonance, 2019, 36(2): 164-171.

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探测应答调控蛋白PhoB_N^F20D自由态中存在的Pre-Active构象

Visualizing the Pre-Active Conformation of Response Regulator PhoB_N^F20D in Its apo State

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探测应答调控蛋白PhoBNF20D自由态中存在的Pre-Active构象

Visualizing the Pre-Active Conformation of Response Regulator PhoBNF20D in Its apo State

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探测应答调控蛋白PhoB_N^F20D自由态中存在的Pre-Active构象

Visualizing the Pre-Active Conformation of Response Regulator PhoB_N^F20D in Its apo State