From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology.
From the Chinese Academy of Sciences Key Laboratory of Synthetic Biology, the State Key Laboratory of Genetic Engineering, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
J Biol Chem. 2014 May 30;289(22):15413-25. doi: 10.1074/jbc.M113.543504. Epub 2014 Apr 14.
The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp(50) residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called "phosphorylation pocket" is not conserved, with its space being occupied by an Arg(52) from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp(50) with the highly conserved Arg(52) and Thr(9) in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation.
放线菌 OmpR/PhoB 亚家族蛋白 GlnR 是一种孤儿应答调节蛋白，它全局协调与氮代谢相关的基因表达。生化和遗传分析表明，来自地中海分枝杆菌的功能性 GlnR 在 N 端受体结构域的潜在磷酸化天冬氨酸（Asp）残基 50 处未发生磷酸化。该受体结构域的晶体结构表明，它通过高度类似于磷酸化典型应答调节蛋白的 α4-β5-α5 二聚体界面形成同源二聚体，而所谓的“磷酸化口袋”没有保守性，其空间被来自β3-α3 环的精氨酸（Arg）52 占据。体外和体内实验均证实，GlnR 通过其受体结构域形成功能性同源二聚体，并表明 GlnR 受体结构域中天冬氨酸（Asp）50 与高度保守的精氨酸（Arg）52 和苏氨酸（Thr）9 的电荷相互作用对于维持同源二聚体的适当构象可能至关重要，分子动力学模拟野生型 GlnR 与缺陷突变体 GlnR（D50A）的结果也支持了这一模型。该模型得到了总缺陷 GlnR（R52A/T9A）双突变体与 GlnR 单个突变体（即 D50N、D50E、R52A 和 T9A）之间明显表型差异的支持，尽管它们的 DNA 结合能力与野生型相比有所减弱，但对 GlnR 体内二聚化和生理功能的影响较小。通过整合模型链霉菌和致病性结核分枝杆菌 GlnR 的支持数据，我们得出结论，放线菌 GlnR 是不典型的，其未磷酸化的保守天冬氨酸残基参与关键的精氨酸/天冬氨酸/苏氨酸电荷相互作用，这对于维持具有生物学活性的同源二聚体构象至关重要。
