{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T16:11:05Z","timestamp":1753891865858,"version":"3.41.2"},"reference-count":48,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,9,3]],"date-time":"2024-09-03T00:00:00Z","timestamp":1725321600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Neurosci."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>The neuronal nitric oxide synthase (nNOS) subtype of nitric oxide synthase (NOS) is an enzyme required for learning and memory. Overactivation of nNOS can lead to oxidative\/nitrite stress, which is complicit in the pathophysiology of various neurological and psychiatric disorders. Previous studies have shown that calmodulin (CaM) forms complexes with Ca<jats:sup>2+<\/jats:sup> and binds to the calmodulin-binding domain (CaMBD) of nNOS, thereby upregulating its catalytic activity in hippocampal neurons. To date, there has been no explanation for the non-covalent interactions in the CaMBD-CaM binding structure model of nNOS.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>In this study, we aimed to investigate the intrinsic factors involved in the binding of CaM to NOS-CaMBD and designed interfering peptides based on the N0 peptide structure of the original nNOS-CaMBD sequence: N1 (obtained from the L734F mutation), N2 (obtained from the F731Y and F740Y mutations), and N3 (obtained from the F731L, V738L, and F740L mutations). We employed homology modeling to construct six CaM-peptide complex models, aiming to elucidate the roles of key amino acid residues within the N0 peptide in its interaction with CaM by means of molecular dynamics simulations. The effect of the peptides on the activation and release of NO by nNOS in neurons was assessed using murine primary neuronal cells.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>When measuring neuronal NO content, it was found that adding N2 and N3 to cultivated neurons significantly increased nNOS activity, leading to the increased NO production. We found that interfering peptides could stably bind to CaM. Among them, N2 and CaM exhibited the strongest binding ability, indicating that the polarized benzene ring significantly enhanced the binding between nNOS-CaMBD and CaM. Conversely, the binding ability between N0 and CaM was the weakest, as they exhibited the worst polar contact, weakest hydrogen bonding, and the lowest binding free energy. The simulation results also highlighted several important amino acid residues: The K76 of CaM plays an important role in polar contact and hydrogen bonding formation, the L734 residue suppressed model flexibility to a certain extent and had an adverse effect on the overall binding free energy of the model. These results, compared with the results of cellular NO content, a preliminary verification of the antagonistic competitive mechanism between CaM allosteric activation of nNOS and SUMOylation hyperactivation was performed.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>In summary, this study explored the ability and mode of action of key residues in nNOS-CaMBD on the binding of interfering peptides to CaM, thereby providing new structural perspectives for the activation of nNOS by CaM and recommendations for drug design targeting the specific inhibition of nNOS.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fnmol.2024.1461272","type":"journal-article","created":{"date-parts":[[2024,9,3]],"date-time":"2024-09-03T05:12:00Z","timestamp":1725340320000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Polarized benzene rings can promote the interaction between CaM and the CaMBD region of nNOS"],"prefix":"10.3389","volume":"17","author":[{"given":"Wei","family":"Wan","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2024,9,3]]},"reference":[{"key":"ref1","doi-asserted-by":"crossref","first-page":"32047","DOI":"10.1016\/S0021-9258(18)31597-7","article-title":"Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer","volume":"269","author":"Abu-Soud","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1042\/0264-6021:3570593","article-title":"Nitric oxide synthases: structure, function and inhibition","volume":"357","author":"Alderton","year":"2001","journal-title":"Biochem. J."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"4813","DOI":"10.1242\/jcs.154229","article-title":"The role of nNOS and PGC-1\u03b1 in skeletal muscle cells","volume":"127","author":"Baldelli","year":"2014","journal-title":"J. Cell Sci."},{"key":"ref4","doi-asserted-by":"crossref","first-page":"10976","DOI":"10.1016\/S0021-9258(19)49862-1","article-title":"Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium\/calmodulin protein kinase; identification of flavin and calmodulin binding sites","volume":"267","author":"Bredt","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"714","DOI":"10.1038\/351714a0","article-title":"Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase","volume":"351","author":"Bredt","year":"1991","journal-title":"Nature"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"682","DOI":"10.1073\/pnas.87.2.682","article-title":"Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme","volume":"87","author":"Bredt","year":"1990","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"volume-title":"Amber 2024","year":"2024","author":"Case","key":"ref7"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"206","DOI":"10.3389\/fphys.2016.00206","article-title":"Neuronal nitric oxide synthase in vascular physiology and diseases","volume":"7","author":"Costa","year":"2016","journal-title":"Front. Physiol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"18","DOI":"10.1089\/ars.2018.7669","article-title":"Activity-induced SUMOylation of neuronal nitric oxide synthase is associated with plasticity of synaptic transmission and extracellular signal-regulated kinase 1\/2 signaling","volume":"32","author":"Du","year":"2020","journal-title":"Antioxid. Redox Signal."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"3891","DOI":"10.1021\/acs.jcim.1c00203","article-title":"AutoDock Vina 1.2.0: new docking methods, expanded force field, and Python bindings","volume":"61","author":"Eberhardt","year":"2021","journal-title":"J. Chem. Inf. Model."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"2126","DOI":"10.1107\/s0907444904019158","article-title":"Coot: model-building tools for molecular graphics","volume":"60","author":"Emsley","year":"2004","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"258","DOI":"10.3389\/fneur.2018.00258","article-title":"Nitric oxide synthase inhibition as a neuroprotective strategy following hypoxic-ischemic encephalopathy: evidence from animal studies","volume":"9","author":"Favi\u00e9","year":"2018","journal-title":"Front. Neurol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1016\/0968-0004(87)90146-0","article-title":"The hydrogen bond in molecular recognition","volume":"12","author":"Fersht","year":"1987","journal-title":"Trends Biochem. Sci."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"1121","DOI":"10.1161\/01.hyp.23.6.1121","article-title":"Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions","volume":"23","author":"F\u00f6rstermann","year":"1994","journal-title":"Hypertension"},{"volume-title":"Gaussian 16 Rev. C.01","year":"2016","author":"Frisch","key":"ref15"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1016\/s0003-9861(03)00009-2","article-title":"Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase","volume":"412","author":"Guan","year":"2003","journal-title":"Arch. Biochem. Biophys."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"101","DOI":"10.1038\/nm.3019","article-title":"Activation of calcium signaling through Trpv1 by nNOS and peroxynitrite as a key trigger of skeletal muscle hypertrophy","volume":"19","author":"Ito","year":"2013","journal-title":"Nat. Med."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"105","DOI":"10.3389\/fncel.2015.00105","article-title":"Aged neuronal nitric oxide knockout mice show preserved olfactory learning in both social recognition and odor-conditioning tasks","volume":"9","author":"James","year":"2015","journal-title":"Front. Cell. Neurosci."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1016\/j.neuroscience.2010.11.005","article-title":"Long-term memory of visually cued fear conditioning: roles of the neuronal nitric oxide synthase gene and cyclic AMP response element-binding protein","volume":"174","author":"Kelley","year":"2011","journal-title":"Neuroscience"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"371","DOI":"10.1101\/lm.1329209","article-title":"Impairments in fear conditioning in mice lacking the nNOS gene","volume":"16","author":"Kelley","year":"2009","journal-title":"Learn. Mem."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"e54834","DOI":"10.1371\/journal.pone.0054834","article-title":"A novel trans conformation of ligand-free calmodulin","volume":"8","author":"Kumar","year":"2013","journal-title":"PLoS One"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"2295","DOI":"10.1007\/s00726-014-1795-y","article-title":"The many structural faces of calmodulin: a multitasking molecular jackknife","volume":"46","author":"Kursula","year":"2014","journal-title":"Amino Acids"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"14860","DOI":"10.1021\/acsnano.2c05756","article-title":"Treating multi-drug-resistant bacterial infections by functionalized Nano-bismuth sulfide through the synergy of immunotherapy and Bacteria-sensitive phototherapy","volume":"16","author":"Li","year":"2022","journal-title":"ACS Nano"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"293","DOI":"10.1016\/j.jinorgbio.2004.10.016","article-title":"Structure-function studies on nitric oxide synthases","volume":"99","author":"Li","year":"2005","journal-title":"J. Inorg. Biochem."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"1251","DOI":"10.1016\/s0006-291x(88)81362-7","article-title":"L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation","volume":"153","author":"Palmer","year":"1988","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"23349","DOI":"10.1074\/jbc.M100687200","article-title":"Calmodulin activates intersubunit electron transfer in the neuronal nitric-oxide synthase dimer","volume":"276","author":"Panda","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"482","DOI":"10.1101\/lm.031450.113","article-title":"Neuronal nitric-oxide synthase deficiency impairs the long-term memory of olfactory fear learning and increases odor generalization","volume":"20","author":"Pavesi","year":"2013","journal-title":"Learn. Mem."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"6081","DOI":"10.1063\/1.460447","article-title":"A complete basis set model chemistry. II. Open-shell systems and the total energies of the first-row atoms","volume":"94","author":"Petersson","year":"1991","journal-title":"J. Chem. Phys."},{"key":"ref29","doi-asserted-by":"crossref","first-page":"2193","DOI":"10.1063\/1.455064","article-title":"A complete basis set model chemistry. I. The total energies of closed-shell atoms and hydrides of the first-row elements","volume":"89","author":"Petersson","year":"1988","journal-title":"J. Chem. Phys."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"299","DOI":"10.1007\/s12104-015-9596-0","article-title":"Chemical shift perturbations induced by residue specific mutations of CaM interacting with NOS peptides","volume":"9","author":"Piazza","year":"2015","journal-title":"Biomol. NMR Assign."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1016\/j.niox.2016.11.004","article-title":"Nitric oxide synthase and structure-based inhibitor design","volume":"63","author":"Poulos","year":"2017","journal-title":"Nitric Oxide"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"3859","DOI":"10.1093\/cercor\/bhaa002","article-title":"Disrupting nNOS-PSD95 interaction improves neurological and cognitive recoveries after traumatic brain injury","volume":"30","author":"Qu","year":"2020","journal-title":"Cereb. Cortex"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"976","DOI":"10.1002\/jcc.1058","article-title":"6-31G* basis set for third-row atoms","volume":"22","author":"Rassolov","year":"2001","journal-title":"J. Comput. Chem."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"23111","DOI":"10.1074\/jbc.M603671200","article-title":"Electron transfer by neuronal nitric-oxide synthase is regulated by concerted interaction of calmodulin and two intrinsic regulatory elements","volume":"281","author":"Roman","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"94","DOI":"10.3389\/fmolb.2019.00094","article-title":"Modeling electrostatic force in protein-protein recognition","volume":"6","author":"Shashikala","year":"2019","journal-title":"Front. Mol. Biosci."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"2071","DOI":"10.1021\/acs.jctc.8b01022","article-title":"Evaluation of predicted protein-protein complexes by binding free energy simulations","volume":"15","author":"Siebenmorgen","year":"2019","journal-title":"J. Chem. Theory Comput."},{"key":"ref37","doi-asserted-by":"crossref","first-page":"11623","DOI":"10.1021\/j100096a001","article-title":"Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields","volume":"98","author":"Stephens","year":"1994","journal-title":"J. Phys. Chem."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"528","DOI":"10.1021\/acs.jctc.9b00591","article-title":"ff19SB: amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution","volume":"16","author":"Tian","year":"2020","journal-title":"J. Chem. Theory Comput."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"4243","DOI":"10.3390\/ijms23084243","article-title":"Roles of nitric oxide in brain ischemia and reperfusion","volume":"23","author":"Wang","year":"2022","journal-title":"Int. J. Mol. Sci."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"535494","DOI":"10.3389\/fnmol.2020.535494","article-title":"The molecular simulation study of nNOS activation induced by the interaction between its calmodulin-binding domain and SUMO1","volume":"13","author":"Wang","year":"2020","journal-title":"Front. Mol. Neurosci."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"4883","DOI":"10.1080\/07391102.2019.1692073","article-title":"Electrostatic interactions in molecular recognition of intrinsically disordered proteins","volume":"38","author":"Yang","year":"2020","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1016\/j.neuroscience.2013.06.012","article-title":"Neuroprotection of S-nitrosoglutathione against ischemic injury by down-regulating Fas S-nitrosylation and downstream signaling","volume":"248","author":"Yin","year":"2013","journal-title":"Neuroscience"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"1120","DOI":"10.1016\/j.neuroscience.2008.03.061","article-title":"Coupling between neuronal nitric oxide synthase and glutamate receptor 6-mediated c-Jun N-terminal kinase signaling pathway via S-nitrosylation contributes to ischemia neuronal death","volume":"155","author":"Yu","year":"2008","journal-title":"Neuroscience"},{"key":"ref45","doi-asserted-by":"publisher","first-page":"811","DOI":"10.1021\/acs.jcim.5b00687","article-title":"VFFDT: a new software for preparing AMBER force field parameters for metal-containing molecular systems","volume":"56","author":"Zheng","year":"2016","journal-title":"J. Chem. Inf. Model."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"1439","DOI":"10.1038\/nm.2245","article-title":"Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95","volume":"16","author":"Zhou","year":"2010","journal-title":"Nat. Med."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"1691","DOI":"10.1021\/acs.chemrev.7b00305","article-title":"Electrostatic interactions in protein structure, folding, binding, and condensation","volume":"118","author":"Zhou","year":"2018","journal-title":"Chem. Rev."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1016\/j.niox.2009.03.001","article-title":"Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications","volume":"20","author":"Zhou","year":"2009","journal-title":"Nitric Oxide"}],"container-title":["Frontiers in Molecular Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2024.1461272\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,9,3]],"date-time":"2024-09-03T05:12:07Z","timestamp":1725340327000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2024.1461272\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,3]]},"references-count":48,"alternative-id":["10.3389\/fnmol.2024.1461272"],"URL":"https:\/\/doi.org\/10.3389\/fnmol.2024.1461272","relation":{},"ISSN":["1662-5099"],"issn-type":[{"type":"electronic","value":"1662-5099"}],"subject":[],"published":{"date-parts":[[2024,9,3]]},"article-number":"1461272"}}