Neira José L, Hornos Felipe, Cozza Concetta, Cámara-Artigas Ana, Abián Olga, Velázquez-Campoy Adrián
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante, Spain; Instituto de Biocomputación y Física de Sistemas Complejos, Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, Spain.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante, Spain.
Arch Biochem Biophys. 2018 Feb 1;639:26-37. doi: 10.1016/j.abb.2017.12.017. Epub 2017 Dec 27.
The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPr, and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPr was 2 μM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPr in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPr, HPr, comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPr was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPr. This implies that specific fragments of Rsd and HPr can be used to interfere with other protein-protein interactions (PPIs) of each other protein.
磷酸转移酶系统（PTS）控制着细菌中糖类的优先利用，并且还参与其他过程，如趋化作用。它由一个蛋白质级联反应构成，其中前两种蛋白质是通用的（即EI和HPr），其他的是糖特异性通透酶。Rsd蛋白特异性结合RNA聚合酶（RNAP）的σ因子。我们首先表征了大肠杆菌Rsd的构象稳定性。其次，我们通过使用源自每种蛋白质的片段，描绘了天蓝色链霉菌、HPr和大肠杆菌Rsd的结合区域。为此，我们使用了几种生物物理探针，即荧光、圆二色性（CD）、核磁共振（NMR）、等温滴定量热法（ITC）和生物层干涉术（BLI）。Rsd在25°C时的解折叠自由能为15千卡/摩尔，在pH 6.5时的热变性中点为103°C。Rsd与HPr之间的亲和力为2μM。有趣的是，包含Rsd的第三（RsdH3）和第四（RsdH4）螺旋的分离螺旋肽也以特定方式与HPr相互作用，并且亲和力与整个Rsd相似。此外，包含活性位点His15的HPr分离肽HPr，也以相似的亲和力与完整的Rsd结合。因此，Rsd与HPr之间的结合受到Rsd的H3和H4两个螺旋以及HPr活性位点周围区域的调节。这意味着Rsd和HPr的特定片段可用于干扰彼此蛋白质的其他蛋白质 - 蛋白质相互作用（PPI）。
