Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
Biochemistry. 2011 Nov 1;50(43):9212-24. doi: 10.1021/bi201226e. Epub 2011 Oct 7.
HetR, master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120, stimulates heterocyst differentiation via transcriptional autoregulation and is negatively regulated by PatS and HetN, both of which contain the active pentapeptide RGSGR. However, the direct targets of PatS and HetN remain uncertain. Here, we report experimental evidence for direct binding between HetR and the C-terminal RGSGR pentapeptide, PatS-5. Strains with a hetR allele coding for conservative substitutions at residues 250-256 had altered patterns of heterocysts and, in some cases, reduced sensitivity to PatS-5. Cysteine scanning mutagenesis coupled with electron paramagnetic resonance (EPR) spectroscopy showed quenching of spin label motion at HetR amino acid 252 upon titration with PatS-5, indicating direct binding of PatS-5 to HetR. Gel shift assays indicated that PatS-5 disrupted binding of HetR to a 29 base pair inverted-repeat-containing DNA sequence upstream from hetP. Double electron-electron resonance EPR experiments confirmed that HetR existed as a dimer in solution and indicated that PatS-5 bound to HetR without disrupting the dimer form of HetR. Isothermal titration calorimetry experiments corroborated direct binding of PatS-5 to HetR with a K(d) of 227 nM and a 1:1 stoichiometry. Taken together, these results indicated that PatS-5 disrupted HetR binding to DNA through a direct HetR/PatS interaction. PatS-5 appeared to either bind in the vicinity of HetR amino acid L252 or, alternately, to bind in a remote site that leads to constrained motion of this amino acid via an allosteric effect or change in tertiary structure.
HetR 是丝状蓝藻鱼腥藻 PCC 7120 中异形胞分化的主要调控因子，通过转录自调控刺激异形胞分化，同时受到 PatS 和 HetN 的负调控，这两者都含有活性五肽 RGSGR。然而，PatS 和 HetN 的直接靶标仍然不确定。在这里，我们报告了 HetR 与 C 末端 RGSGR 五肽 PatS-5 之间直接结合的实验证据。HetR 等位基因编码 250-256 残基保守取代的菌株具有改变的异形胞模式，在某些情况下，对 PatS-5 的敏感性降低。与电子顺磁共振 (EPR) 光谱相结合的半胱氨酸扫描突变表明，用 PatS-5 滴定时，HetR 氨基酸 252 的自旋标记运动被猝灭，表明 PatS-5 直接与 HetR 结合。凝胶移位分析表明，PatS-5 破坏了 HetR 与 hetP 上游含 29 个碱基对反向重复的 DNA 序列的结合。双电子-电子共振 EPR 实验证实 HetR 以二聚体形式存在于溶液中，并表明 PatS-5 与 HetR 结合而不破坏 HetR 的二聚体形式。等温滴定量热法实验证实了 PatS-5 与 HetR 的直接结合，K(d)为 227 nM，为 1:1 化学计量比。综上所述，这些结果表明，PatS-5 通过直接的 HetR/PatS 相互作用破坏了 HetR 与 DNA 的结合。PatS-5 似乎要么结合在 HetR 氨基酸 L252 的附近，要么结合在一个远程位点，通过变构效应或三级结构的改变导致该氨基酸的受限运动。
