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. 15N/13C-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.
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - 26 Aug 2017|
Bibliographical noteFunding Information:
This work was supported by Mid-career Researcher Program (NRF-2017R1A2B2008483 to WL) and the Basic Science Research Program (NRF-518 2016R1A6A3A04010213n to J. H. Yun) through the National Research Foundation of Korea funded by the Ministry of Education. This work was partially supported by the Translational Research Center for Protein Function Control (Grant Number 2016R1A5A1004694).
© 2017 Elsevier Inc.
- NMR (Nuclear magnetic resonance)
- NOX1 (NADPH Oxidase 1)
- Noxa1 (NADPH Oxidase activator 1)
- Noxo1 (NADPH Oxidase organizer 1)
- PRR (Proline rich region)
- ROS (Reactive oxygen species)