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- PDB-8fgj: Structure of human neuronal nitric oxide synthase R354A/G357D mut... -

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Basic information

Entry
Database: PDB / ID: 8fgj
TitleStructure of human neuronal nitric oxide synthase R354A/G357D mutant heme domain in complex with 6-(5-(2-(dimethylamino)ethyl)-2,3-difluorophenethyl)-4-methoxypyridin-2-amine
ComponentsNitric oxide synthase, brain
KeywordsOXIDOREDUCTASE / nitric oxide synthase inhibitor / heme enzyme
Function / homology
Function and homology information


negative regulation of calcium ion transport into cytosol / myoblast fusion / Nitric oxide stimulates guanylate cyclase / ROS and RNS production in phagocytes / negative regulation of hydrolase activity / tetrahydrobiopterin binding / arginine binding / regulation of calcium ion transmembrane transport via high voltage-gated calcium channel / retrograde trans-synaptic signaling by nitric oxide / positive regulation of sodium ion transmembrane transport ...negative regulation of calcium ion transport into cytosol / myoblast fusion / Nitric oxide stimulates guanylate cyclase / ROS and RNS production in phagocytes / negative regulation of hydrolase activity / tetrahydrobiopterin binding / arginine binding / regulation of calcium ion transmembrane transport via high voltage-gated calcium channel / retrograde trans-synaptic signaling by nitric oxide / positive regulation of sodium ion transmembrane transport / positive regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / peptidyl-cysteine S-nitrosylation / cadmium ion binding / positive regulation of the force of heart contraction / negative regulation of potassium ion transport / negative regulation of calcium ion transport / negative regulation of serotonin uptake / sodium channel regulator activity / striated muscle contraction / nitric-oxide synthase (NADPH) / regulation of cardiac muscle contraction / multicellular organismal response to stress / xenobiotic catabolic process / nitric-oxide synthase activity / nitric oxide mediated signal transduction / arginine catabolic process / regulation of sodium ion transport / regulation of ryanodine-sensitive calcium-release channel activity / Ion homeostasis / photoreceptor inner segment / nitric oxide biosynthetic process / negative regulation of blood pressure / response to hormone / cell redox homeostasis / sarcoplasmic reticulum / muscle contraction / cell periphery / sarcolemma / cellular response to growth factor stimulus / vasodilation / calcium-dependent protein binding / FMN binding / flavin adenine dinucleotide binding / positive regulation of peptidyl-serine phosphorylation / NADP binding / response to heat / scaffold protein binding / transmembrane transporter binding / response to lipopolysaccharide / dendritic spine / postsynaptic density / response to hypoxia / cytoskeleton / calmodulin binding / membrane raft / synapse / heme binding / perinuclear region of cytoplasm / positive regulation of DNA-templated transcription / positive regulation of transcription by RNA polymerase II / protein-containing complex / mitochondrion / nucleoplasm / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Nitric-oxide synthase, eukaryote / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 3 superfamily / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, oxygenase domain / Nitric oxide synthase (NOS) signature. / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily ...Nitric-oxide synthase, eukaryote / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 3 superfamily / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, oxygenase domain / Nitric oxide synthase (NOS) signature. / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / FAD binding domain / Flavodoxin-like / Flavoprotein pyridine nucleotide cytochrome reductase / Flavodoxin / Flavodoxin-like domain profile. / Flavodoxin/nitric oxide synthase / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / PDZ domain / Flavoprotein-like superfamily / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily
Similarity search - Domain/homology
5,6,7,8-TETRAHYDROBIOPTERIN / PROTOPORPHYRIN IX CONTAINING FE / Chem-XVZ / Nitric oxide synthase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.15 Å
AuthorsLi, H. / Poulos, T.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM57353 United States
CitationJournal: J.Med.Chem. / Year: 2023
Title: Potent, Selective, and Membrane Permeable 2-Amino-4-Substituted Pyridine-Based Neuronal Nitric Oxide Synthase Inhibitors.
Authors: Vasu, D. / Do, H.T. / Li, H. / Hardy, C.D. / Awasthi, A. / Poulos, T.L. / Silverman, R.B.
History
DepositionDec 12, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nitric oxide synthase, brain
B: Nitric oxide synthase, brain
C: Nitric oxide synthase, brain
D: Nitric oxide synthase, brain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,84926
Polymers195,9874
Non-polymers5,86222
Water8,287460
1
A: Nitric oxide synthase, brain
B: Nitric oxide synthase, brain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,92513
Polymers97,9942
Non-polymers2,93111
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9870 Å2
ΔGint-82 kcal/mol
Surface area33910 Å2
MethodPISA
2
C: Nitric oxide synthase, brain
D: Nitric oxide synthase, brain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,92513
Polymers97,9942
Non-polymers2,93111
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9870 Å2
ΔGint-83 kcal/mol
Surface area34510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.170, 121.407, 164.651
Angle α, β, γ (deg.)90.00, 90.04, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Nitric oxide synthase, brain / / Constitutive NOS / NC-NOS / NOS type I / Neuronal NOS / nNOS / Peptidyl-cysteine S-nitrosylase NOS1 / bNOS


Mass: 48996.781 Da / Num. of mol.: 4 / Mutation: R354A, G357D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NOS1 / Organ: brain / Plasmid: pCWori / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P29475, nitric-oxide synthase (NADPH)

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Non-polymers , 6 types, 482 molecules

#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical
ChemComp-H4B / 5,6,7,8-TETRAHYDROBIOPTERIN / Tetrahydrobiopterin


Mass: 241.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H15N5O3 / Comment: neurotransmitter*YM
#4: Chemical
ChemComp-XVZ / 6-{2-[5-(2-aminoethyl)-2,3-difluorophenyl]ethyl}-4-methylpyridin-2-amine


Mass: 291.339 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C16H19F2N3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 460 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.74 Å3/Da / Density % sol: 55.2 % / Description: plates
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 8% PEG3350 35mM citric acid 65mM Bis-Tris-Propane 10% glycerol 5mM TCEP

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 2, 2021 / Details: mirrors
RadiationMonochromator: DOUBLE CRYSTAL SI(III) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.15→39.57 Å / Num. obs: 109389 / % possible obs: 98.3 % / Redundancy: 4.6 % / CC1/2: 0.997 / Rmerge(I) obs: 0.124 / Rpim(I) all: 0.064 / Rrim(I) all: 0.14 / Net I/σ(I): 7.3 / Num. measured all: 506986
Reflection shellResolution: 2.15→2.19 Å / Redundancy: 4.7 % / Rmerge(I) obs: 2.909 / Num. unique obs: 5490 / CC1/2: 0.407 / Rpim(I) all: 1.473 / Rrim(I) all: 3.274 / % possible all: 99

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Processing

Software
NameVersionClassification
Aimless0.7.4data scaling
PHENIX1.11.1_2575refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 7TS7

7ts7


Resolution: 2.15→39.566 Å / SU ML: 0.31 / Cross valid method: THROUGHOUT / σ(F): 0 / Phase error: 35.57 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2609 10642 5.02 %random
Rwork0.1976 ---
obs0.2008 105647 96.48 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.15→39.566 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13443 0 404 460 14307
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00914321
X-RAY DIFFRACTIONf_angle_d1.02819491
X-RAY DIFFRACTIONf_dihedral_angle_d18.4938341
X-RAY DIFFRACTIONf_chiral_restr0.0552007
X-RAY DIFFRACTIONf_plane_restr0.0072453
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.15-2.17440.40613670.32076191X-RAY DIFFRACTION88
2.1744-2.20.36663080.31876183X-RAY DIFFRACTION89
2.2-2.22680.36633260.31056189X-RAY DIFFRACTION91
2.2268-2.2550.393400.32366313X-RAY DIFFRACTION89
2.255-2.28470.38713670.31096327X-RAY DIFFRACTION92
2.2847-2.3160.36473740.29756626X-RAY DIFFRACTION96
2.316-2.34910.34573690.29026805X-RAY DIFFRACTION97
2.3491-2.38410.32813920.27516810X-RAY DIFFRACTION97
2.3841-2.42140.32333210.27036733X-RAY DIFFRACTION98
2.4214-2.46110.39143930.29046730X-RAY DIFFRACTION96
2.4611-2.50350.32583500.28986640X-RAY DIFFRACTION95
2.5035-2.5490.29483650.26496790X-RAY DIFFRACTION98
2.549-2.5980.31113810.25666837X-RAY DIFFRACTION98
2.598-2.65110.31893440.26676818X-RAY DIFFRACTION99
2.6511-2.70870.29823510.2696962X-RAY DIFFRACTION99
2.7087-2.77170.36423430.27976868X-RAY DIFFRACTION99
2.7717-2.8410.34063340.25746854X-RAY DIFFRACTION99
2.841-2.91780.30883770.23246892X-RAY DIFFRACTION98
2.9178-3.00360.26533080.2356898X-RAY DIFFRACTION99
3.0036-3.10050.32443590.24166924X-RAY DIFFRACTION98
3.1005-3.21130.34513180.24546573X-RAY DIFFRACTION95
3.2113-3.33980.31063610.22586745X-RAY DIFFRACTION97
3.3398-3.49170.28464240.21246944X-RAY DIFFRACTION99
3.4917-3.67570.23943470.17666884X-RAY DIFFRACTION99
3.6757-3.90580.24783930.1716902X-RAY DIFFRACTION99
3.9058-4.2070.22213610.15736804X-RAY DIFFRACTION99
4.207-4.62980.19243710.14136774X-RAY DIFFRACTION98
4.6298-5.29840.18073050.13056795X-RAY DIFFRACTION96
5.2984-6.67030.22553630.15826901X-RAY DIFFRACTION99
6.6703-39.5660.19053300.15166750X-RAY DIFFRACTION97
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.6926-0.3089-0.17141.124-0.33556.7414-0.08210.1734-0.0244-0.0956-0.0965-0.06670.2855-0.23650.15310.3784-0.0701-0.03350.5753-0.03020.2986-11.46427.421-40.926
21.2173-0.27340.03391.3315-0.40573.08410.00070.0053-0.0849-0.0921-0.06010.0854-0.0404-0.31420.05260.2572-0.0419-0.05550.3722-0.06610.256-12.62326.856-3.574
31.23560.23620.07931.4007-0.44783.09490.0108-0.03310.12560.1103-0.05310.1020.0084-0.28180.03560.24210.04380.04180.3462-0.06510.2519-38.602-24.53-78.736
40.87650.239-0.01671.2036-0.24726.8773-0.0786-0.18430.04530.0817-0.0821-0.0682-0.2634-0.1950.13690.38170.06470.00590.566-0.02620.2863-37.508-25.211-41.434
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 304:722 )A304 - 722
2X-RAY DIFFRACTION2( CHAIN B AND RESID 304:722 )B304 - 722
3X-RAY DIFFRACTION3( CHAIN C AND RESID 303:722 )C303 - 722
4X-RAY DIFFRACTION4( CHAIN D AND RESID 304:722 )D304 - 722

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