# FNR-like unit interacts with C-terminal related residues trigger nNOS reductase domain conformational flexibility change

## TL;DR

This study explores how specific residues in the nNOS enzyme affect cofactor binding and enzyme activity, offering insights for better inhibitors to treat neurological disorders.

## Contribution

The study identifies novel residues and their roles in stabilizing cofactors in the FNR-like unit of nNOS, providing new targets for drug development.

## Key findings

- R1400, R1284, R1173, and F1395 are key residues stabilizing cofactor binding in the FNR-like unit.
- RMSD and RMSF analyses confirm structural stability and low flexibility of these residues.
- In silico mutagenesis shows these residues significantly influence the energy landscape of the domain.

## Abstract

The reductase domain of neuronal nitric oxide synthase (nNOS) is essential for nitric oxide (NO) production in the mammalian nervous system. Excessive NO contributes to neurological disorders, including ischemic stroke, highlighting the need to better understand the structural dynamics of this domain. The FNR- like unit within the reductase domain stabilizes the cofactors NADP(H), FAD, and FMN, which are critical for NO synthesis. However, the dynamic interactions between these cofactors and key residues remain poorly characterized, limiting the ability of current nNOS inhibitors to normalize enzyme activity in cerebral ischemia–reperfusion injury.

In this study, we evaluated the effects of widely used nNOS inhibitors, including spermidine (Spe) and L-NMMA, on nNOS activity using cellular NO assays and Western blot analysis. To investigate the structural basis of this resistance, we constructed three molecular models representing distinct redox states of NADP(H), FAD, and FMN, and derived new parameters for these cofactors (oxidized and reduced forms) using the def2-TZVP basis set.

While those inhibitors showed some therapeutic benefit in ischemia–reperfusion injury, they failed to suppress nNOS activity to physiological levels. RMSD analysis confirmed conformational stability after ~1.0 μs of simulation. Hydrogen bond and polar contact analyses identified R1400, R1284, R1173, and F1395 as key residues stabilizing cofactor binding in the FNR-like unit. RMSF analysis revealed low flexibility of these residues, supporting structural integrity. Correlation and free energy calculations further demonstrated their critical contributions to the energy landscape. In silico site-directed mutagenesis of these residues induced significant free energy changes, confirming their role in modulating domain dynamics. Notably, R1400 and F1395, previously associated with the calmodulin-binding domain, influence conformational flexibility, while R1173 represents a novel interaction hotspot distinct from R1284.

These results provide detailed insights into the dynamic mechanisms of the FNR-like unit and identify promising targets for the development of improved nNOS inhibitors to control excessive NO production in neurological disorders such as ischemic stroke.