探测应答调控蛋白PhoBNF20D自由态中存在的Pre-Active构象

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Mg^2+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PhoBN^F20D作为模型,1H-15NHSQC和19F NMR谱图显示,自由态PhoBN^F20D在溶液中存在pre-active构象,BeF3^-可以稳定这种活性构象,而且Mg^2+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PhoBN^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PhoBN^F20D存在两态交换.

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

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^-.