Shrestha Pravesh, Yun Ji-Hye, Ko Yoon-Joo, Kim Myeongkyu, Bae Yun Soo, Lee Weontae
Structural Biochemistry & Molecular Biophysics Laboratory, Department of Biochemistry, College of Life Sciences & Biotechnology, Yonsei University, Seoul 03722, South Korea.
Structural Biochemistry & Molecular Biophysics Laboratory, Department of Biochemistry, College of Life Sciences & Biotechnology, Yonsei University, Seoul 03722, South Korea.
Biochem Biophys Res Commun. 2017 Aug 26;490(3):594-600. doi: 10.1016/j.bbrc.2017.06.083. Epub 2017 Jun 16.
NOX1 (NADPH oxidase) similar to phagocyte NADPH oxidase, is expressed mainly in the colon epithelium and it is responsible for host defense against microbial infections by generating ROS (reactive oxygen species). NOX1 is activated by two regulatory cytosolic proteins that form a hetero-dimer, Noxo1 (NOX organizer 1) and Noxa1 (NOX activator 1). The interaction between Noxa1 and Noxo1 is critical for activating NOX1. However no structural studies for interaction between Noxa1 and Noxo1 has not been reported till date. Here, we studied the inter-molecular interaction between the SH3 domain of Noxa1 and Noxo1 using pull-down assay and NMR spectroscopy. N/C-labeled SH3 domain of Noxa1 has been purified for hetero-nuclear NMR experiments (HNCACB, CBCACONH, HNCA, HNCO, and HSQC). TALOS analysis using backbone assignment data of the Noxa1 SH3 domain showed that the structure primarily consists of β-sheets. Data from pull-down assay between the Noxo1 and Noxa1 showed that the SH3 domains (Noxa1) is responsible for interaction with Noxo1 C-terminal tail harboring proline rich region (PRR). The concentration-dependent titration of the Noxo1 C-terminal tail to Noxa1 shows that Noxo1 particularly in the RT loop: Q407*, H408, S409, A412*, G414*, E416, D417, L418, and F420; n-Src loop: C430, E431*, V432*, A435, W436, and L437; and terminal region: I447; F448*, F452* and V454 interact with Noxa1. Our results will provide a detailed understanding for interaction between Noxa1 and Noxo1 at the molecular level, providing insights into their cytoplasmic activity-mediated functioning as well as regulatory role of C-terminal tail of Noxo1 in the NOX1 complex.
NOX1（烟酰胺腺嘌呤二核苷酸磷酸氧化酶）与吞噬细胞烟酰胺腺嘌呤二核苷酸磷酸氧化酶相似，主要在结肠上皮中表达，通过产生活性氧（ROS）负责宿主抵御微生物感染。NOX1由两种形成异二聚体的调节性胞质蛋白Noxo1（NOX组织者1）和Noxa1（NOX激活剂1）激活。Noxa1和Noxo1之间的相互作用对于激活NOX1至关重要。然而，迄今为止尚未报道关于Noxa1和Noxo1之间相互作用的结构研究。在这里，我们使用下拉实验和核磁共振光谱研究了Noxa1和Noxo1的SH3结构域之间的分子间相互作用。已纯化N/C标记的Noxa1的SH3结构域用于异核核磁共振实验（HNCACB、CBCACONH、HNCA、HNCO和HSQC）。使用Noxa1 SH3结构域的主链归属数据进行的TALOS分析表明，该结构主要由β折叠组成。Noxo1和Noxa1之间的下拉实验数据表明，SH3结构域（Noxa1）负责与含有富含脯氨酸区域（PRR）的Noxo1 C末端尾巴相互作用。Noxo1 C末端尾巴与Noxa1的浓度依赖性滴定表明，Noxo1特别是在RT环：Q407*、H408、S409、A412*、G414*、E416、D417、L418和F420；n-Src环：C430、E431*、V432*、A435、W436和L437；以及末端区域：I447；F448*、F452*和V454与Noxa1相互作用。我们的结果将在分子水平上提供对Noxa1和Noxo1之间相互作用的详细理解，深入了解它们的细胞质活性介导的功能以及Noxo1 C末端尾巴在NOX1复合物中的调节作用。
