초록/요약

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 Noxal (NOX activator 1). The interaction between Noxal and Noxol is critical for activating NOX1. However no structural studies for interaction between Noxal and Noxol has not been reported till date. Here, we studied the inter-molecular interaction between the SH3 domain of Noxal and Noxol using pull-down assay and NMR spectroscopy.N-15/C-13-labeled SH3 domain of Noxal has been purified for hetero-nuclear NMR experiments (HNCACB, CBCACONH, HNCA, HNCO, and HSQC). TALOS analysis using backbone assignment data of the Noxal SH3 domain showed that the structure primarily consists of 13-sheets. Data from pull-down assay between the Noxol and Noxal showed that the SH3 domains (Noxal) is responsible for interaction with NoxolC-terminal tail harboring proline rich region (PRR). The concentration-dependent titration of the Noxol C-terminal tail to Noxa1 shows that Noxol 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 Noxal and Noxol 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. (C) 2017 Elsevier Inc. All rights reserved.