Abstract
Neuronal nitric oxide synthase (nNOS), an enzyme required for learning and memory, catalyzes L-arginine decomposition during nitric oxide production in mammalian neurons. Over-activation of nNOS leads to oxidative/nitrosative stress, which is part of the pathophysiological process of various neuropsychiatric disorders. Previous experimental studies suggest that nNOS is a target for small ubiquitin-like modifier 1 (SUMO1), and that SUMO1-ylation upregulates nNOS catalytic activity in hippocampal neurons. To date, a comprehensive structural model has not been proposed for nNOS SUMO1-ylation. In this study, our aim was to build in silico models to identify the non-bonded interactions between SUMO1 and the calmodulin binding domain (CaMBD) of nNOS. Using molecular docking and molecular dynamics simulation, we found that SUMO1 modification stabilizes the conformation of nNOS CaMBD, and helps maintain a conformation beneficial for nNOS catalysis. Analysis of the polar contacts and hydrogen bonds, and the root mean square derivation results showed that R726 and R727 of CaMBD formed polar contacts or high occupancy hydrogen bonds with SUMO1. Correlation factor analysis and free energy calculations showed that the W716, L734, F740, M745, and F781 residues were also involved in the SUMO1/CaMBD interaction in an orientation-dependent manner. The potential inhibitor binding pocket of SUMO1, aimed at disrupting SUMO1/CaMBD binding, was detected from the virtual screening results. Our in silico studies revealed that interfering with the non-bonded interactions of SUMO1/CaMBD would blocked nNOS SUMO-ylation and subsequent hyperactivation. This work provides novel structural insight into the functional regulation of nNOS by post-translational SUMO1 modification, and provides suggestions for the design of drugs targeting nNOS hyperactivation.
Highlights
Neuronal nitric oxide synthase, an enzyme constitutively expressed and of high levels in the mammalian brain and skeletal muscle, catalyzes the conversion of L-arginine to nitric oxide
Among the 100 Z-DOCK outputs, the top 15 conformations ranked based on their Z-DOCK scores showed that the calmodulin-binding domain (CaMBD) region of Neuronal nitric oxide synthase (nNOS) was favorable for small ubiquitin-like modifier 1 (SUMO1) docking (Figure 1B); SUMO1 was seen in close contact with CaMBD in 12 out of 15 conformations
We generated three SUMO1/CaMBD binding models and performed polar contacts analysis, hydrogen bond statistics, root mean square fluctuation (RMSF), correlation factor analysis, and free energy analysis on them. They represent the most stable/detailed binding models for the structural interactions between SUMO1 and nNOS CaMBD, but they point out the spatial characteristics of the nNOS SUMO-ylation site, which provide further insights into the study of protein SUMO-ylation
Summary
Neuronal nitric oxide synthase (nNOS), an enzyme constitutively expressed and of high levels in the mammalian brain and skeletal muscle, catalyzes the conversion of L-arginine to nitric oxide. We have referred to the partial reductase domain as the FAD-binding domain (residues 755–951), since it contains the critical residues for FAD binding. In this nNOS dimer model, the FAD-binding domain in one chain interacts with the heme domain in another chain, and together with the remaining part of the heme domain, it forms the FAD-binding site. This theoretical model partially clarifies certain molecular functions like the synthase activity of nNOS, it lacks structural insight on the effect of covalent modifications on nNOS catalysis
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