Centro de Investigación en Alimentación y Desarrollo A. C., Km 0.6 Carretera a La Victoria, 83304, Hermosillo, Sonora, Mexico.
Appl Microbiol Biotechnol. 2014 Nov;98(22):9399-412. doi: 10.1007/s00253-014-6094-8. Epub 2014 Sep 26.
NprR belongs to the RNPP family of quorum-sensing receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria. In Bacillus thuringiensis (Bt), nprR is located in a transcriptional cassette with nprRB that codes for the precursor of the signaling peptide NprRB. NprR is a transcriptional regulator activated by binding of reimported NprRB; however, several reports suggest that NprR also participates in sporulation but the mechanism is unknown. Our in silico results, based on the structural similarity between NprR from Bt and Spo0F-binding Rap proteins from Bacillus subtilis, suggested that NprR could bind Spo0F to modulate the sporulation phosphorelay in Bt. Deletion of nprR-nprRB cassette from Bt caused a delay in sporulation and defective trigger of the Spo0A∼P-activated genes spoIIA and spoIIIG. The DNA-binding domain of NprR was not necessary for this second function, since truncated NprRΔHTH together with nprRB gene was able to restore the sporulation wild type phenotype in the ΔnprR-nprRB mutant. Fluorescence assays showed direct binding between NprR and Spo0F, supporting that NprR is a bifunctional protein. To understand how the NprR activation by NprRB could result in two different functions, we studied the molecular recognition mechanism between the signaling peptide and the receptor. Using synthetic variants of NprRB, we found that SSKPDIVG displayed the highest affinity (Kd = 7.19 nM) toward the recombinant NprR and demonstrated that recognition involves conformational selection. We propose that the peptide concentration in the cell controls the oligomerization state of the NprR-NprRB complex for switching between its two functions.
NprR 属于 RNPP 家族的群体感应受体，这是一组直接被革兰氏阳性细菌信号寡肽激活的细胞内调节剂。在苏云金芽孢杆菌（Bt）中，nprR 位于一个转录盒中，与编码信号肽 NprRB 前体的 nprRB 一起编码。NprR 是一种转录调节剂，通过再导入的 NprRB 结合而被激活；然而，有几项报告表明，NprR 也参与孢子形成，但机制尚不清楚。我们基于苏云金芽孢杆菌 NprR 与枯草芽孢杆菌 Spo0F 结合 Rap 蛋白之间的结构相似性的计算机模拟结果表明，NprR 可以结合 Spo0F 来调节 Bt 中的孢子形成磷酸接力。从 Bt 中删除 nprR-nprRB 盒会导致孢子形成延迟和 Spo0A∼P 激活基因 spoIIA 和 spoIIIG 的触发缺陷。NprR 的 DNA 结合域对于该第二功能不是必需的，因为截断的 NprRΔHTH 与 nprRB 基因一起能够在 ΔnprR-nprRB 突变体中恢复野生型孢子形成表型。荧光测定表明 NprR 和 Spo0F 之间存在直接结合，支持 NprR 是一种双功能蛋白。为了理解 NprRB 对 NprR 的激活如何导致两种不同的功能，我们研究了信号肽和受体之间的分子识别机制。使用 NprRB 的合成变体，我们发现 SSKPDIVG 对重组 NprR 表现出最高的亲和力（Kd=7.19 nM），并证明识别涉及构象选择。我们提出，细胞内的肽浓度控制 NprR-NprRB 复合物的寡聚状态，从而在其两种功能之间进行切换。
