Table 1.
Selected sites for mutagenesis analysis.
Fig 1.
RaptorX-predicted in silico structure model of human NOX1.
(A) Side view of NOX1 in silico structure model. (B) Extracellular view (bird’s eye view) of NOX1 model. Marine blue represents 6-TM loop domain, golden represents FAD binding domain and light pink for NADPH binding/NOXO1 interacting region.
Fig 2.
Comparison of predicted models of human NOX1 and experimentally resolved models of csNOX5, human DUOX1 and core human NOX2 transmembrane region.
(A) Superimposed view of RaptorX-predicted (in dark blue) and tFold-predicted (in green) human NOX1 model. (B) Superimposed view of RaptorX-predicted (in dark blue) and AlphaFold-predicted (in pink) human NOX1 model. (C) Superimposed view of RaptorX-predicted human NOX1 model (in dark blue) and previously reported csNOX5 model (PDB ID: 5O0T for transmembrane domain, PDB ID: 5O0X for dehydrogenase domain, shown in yellow). (D) Superimposed view of RaptorX-predicted human NOX1 model (in dark blue) and previously reported human DUOX1 model (PDB ID: 7D3E, chain A, shown in orange). Root mean square distance (RMSD) were calculated and shown for structural similarity. (E) Superimposed view of RaptorX-predicted human NOX1 model (in dark blue) and human NOX2 transmembrane region model (PDB ID: 7U8G, chain A, shown in salmon red).
Fig 3.
Binding schemes of the heme group, FAD and NADPH to NOX1.
(A) & (B) Binding poses of heme groups into the 6-TM loop domain of NOX1. (C) Binding of FAD into the C-terminus intracellular FAD binding domain of NOX1. (D) Binding of NADPH into the C-terminus intracellular NADPH binding domain of NOX1. (E) Overview of the heme group, FAD and NADPH binding to NOX1.
Fig 4.
Schematic illustration of electron transfer in NOX1.
The electrons are donated by an NADPH at the NADPH interacting domain of NOX1. A FAD molecule then accepts the two electrons released from the NADPH molecule, and is reduced to FADH2, which further passes two electrons on to the transmembrane heme groups. The electrons are then transported through the 6-TM loop region towards the extracellular part of NOX1. The extracellular region provides a harbor for the final step of ROS generation, where two oxygen molecules encounter two electrons released by the outer-heme group and are reduced into the free radical form of O2•−.
Fig 5.
A ROS response in mutagenesis assays. Data are illustrated as percentages of the positive control. Positive: Wild-type NOX1; Negative: ROS generation in cells transfected with an empty plasmid. Five independent experiments were included for each mutant. B A representative illustration of enzyme kinetics.
Fig 6.
Superimposed binding scheme of the NOX1 inhibitors.
Table 2.
Representative NOX1 inhibitors for docking analysis.
Fig 7.
Binding of NOX1 inhibitors deep into the center of the TM domain of NOX1, showing the binding pockets of (A) GKT137831, (B) GKT136901, (C) ML171, (D) ML090, (E) VAS2870, (F) VAS3947.
Fig 8.
ROS inhibition assay of NOX1 mutants.
(A) ROS generation of NOX1 wild-type and mutants without inhibitor treatment, ROS generation of WT and mutants treated with 10 μM (B) GKT137831, (C) ML171, (D) VAS2870.