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

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Basic information

Entry
Database: PDB / ID: 8fgl
TitleStructure of human neuronal nitric oxide synthase R354A/G357D mutant heme domain in complex with 6-(2,3-difluoro-5-(2-(4-methylpiperazin-1-yl)ethyl)phenethyl)-4-methylpyridin-2-amine
ComponentsNitric oxide synthase, brain
KeywordsOXIDOREDUCTASE / nitric oxide synthase inhibitor / heme enzyme
Function / homology
Function and homology information


positive regulation of membrane repolarization during ventricular cardiac muscle cell action potential / negative regulation of calcium ion transport into cytosol / Nitric oxide stimulates guanylate cyclase / myoblast fusion / ROS and RNS production in phagocytes / negative regulation of hydrolase activity / tetrahydrobiopterin binding / regulation of cardiac muscle contraction by calcium ion signaling / arginine binding / regulation of calcium ion transmembrane transport via high voltage-gated calcium channel ...positive regulation of membrane repolarization during ventricular cardiac muscle cell action potential / negative regulation of calcium ion transport into cytosol / Nitric oxide stimulates guanylate cyclase / myoblast fusion / ROS and RNS production in phagocytes / negative regulation of hydrolase activity / tetrahydrobiopterin binding / regulation of cardiac muscle contraction by calcium ion signaling / arginine binding / regulation of calcium ion transmembrane transport via high voltage-gated calcium channel / 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 / regulation of cardiac muscle contraction / negative regulation of serotonin uptake / calcium channel regulator activity / nitric-oxide synthase (NADPH) / sodium channel regulator activity / regulation of ryanodine-sensitive calcium-release channel activity / nitric oxide mediated signal transduction / nitric-oxide synthase activity / xenobiotic catabolic process / multicellular organismal response to stress / arginine catabolic process / striated muscle contraction / regulation of sodium ion transport / Ion homeostasis / nitric oxide biosynthetic process / negative regulation of blood pressure / photoreceptor inner segment / response to hormone / cell redox homeostasis / sarcoplasmic reticulum / cell periphery / sarcolemma / cellular response to growth factor stimulus / vasodilation / calcium-dependent protein binding / FMN binding / positive regulation of peptidyl-serine phosphorylation / flavin adenine dinucleotide binding / NADP binding / response to heat / scaffold protein binding / transmembrane transporter binding / response to lipopolysaccharide / dendritic spine / postsynaptic density / cytoskeleton / calmodulin binding / response to hypoxia / membrane raft / heme binding / synapse / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / 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, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 3 superfamily / : / Nitric oxide synthase, oxygenase domain / Nitric oxide synthase (NOS) signature. / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding ...Nitric-oxide synthase, eukaryote / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 3 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-XVK / Nitric oxide synthase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 2.1 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

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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)200,75024
Polymers195,1384
Non-polymers5,61220
Water9,332518
1
A: Nitric oxide synthase, brain
B: Nitric oxide synthase, brain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,37512
Polymers97,5692
Non-polymers2,80610
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10060 Å2
ΔGint-85 kcal/mol
Surface area34080 Å2
MethodPISA
2
C: Nitric oxide synthase, brain
D: Nitric oxide synthase, brain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,37512
Polymers97,5692
Non-polymers2,80610
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9920 Å2
ΔGint-83 kcal/mol
Surface area34230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.030, 121.912, 164.655
Angle α, β, γ (deg.)90.000, 90.100, 90.000
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: 48784.496 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, 538 molecules

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


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


Mass: 241.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H15N5O3 / Comment: neurotransmitter*YM
#4: Chemical
ChemComp-XVK / 6-(2-{2,3-difluoro-5-[2-(4-methylpiperazin-1-yl)ethyl]phenyl}ethyl)-4-methylpyridin-2-amine


Mass: 374.471 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H28F2N4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


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


Mass: 18.015 Da / Num. of mol.: 518 / 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: 7.2
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: 0.9795 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jun 14, 2021 / Details: mirrors
RadiationMonochromator: DOUBLE CRYSTAL SI(III) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.1→39.574 Å / Num. obs: 119490 / % possible obs: 99.9 % / Redundancy: 4.6 % / Biso Wilson estimate: 40.44 Å2 / CC1/2: 0.989 / Rmerge(I) obs: 0.237 / Rpim(I) all: 0.116 / Rrim(I) all: 0.265 / Net I/σ(I): 4.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.1-2.144.55.5012682859200.3372.6766.1370.499.8
11.5-39.574.90.06136657490.9960.030.06815.897.7

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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: 2.1→39.574 Å / SU ML: 0.41 / Cross valid method: FREE R-VALUE / σ(F): 0.77 / Phase error: 37.56 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.281 10951 5 %random
Rwork0.2154 208115 --
obs0.2187 112204 92.75 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 163.9 Å2 / Biso mean: 59.392 Å2 / Biso min: 20.58 Å2
Refinement stepCycle: final / Resolution: 2.1→39.574 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13449 0 386 518 14353
Biso mean--55.2 51.63 -
Num. residues----1650
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00914327
X-RAY DIFFRACTIONf_angle_d1.08719505
X-RAY DIFFRACTIONf_chiral_restr0.0552017
X-RAY DIFFRACTIONf_plane_restr0.0082455
X-RAY DIFFRACTIONf_dihedral_angle_d18.658380
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.1-2.12390.54962240.4812401154
2.1239-2.14890.45533060.4308590878
2.1489-2.17510.43223530.4077610482
2.1751-2.20260.42093430.4046658089
2.2026-2.23160.48772240.4519437958
2.2316-2.26210.52683830.4372733196
2.2621-2.29440.47322760.3835515070
2.2944-2.32870.38744030.3414700594
2.3287-2.36510.36123880.3379728896
2.3651-2.40380.35043810.3165709497
2.4038-2.44530.38033960.3283721097
2.4453-2.48970.34993720.3131725096
2.4897-2.53760.33853600.313723097
2.5376-2.58940.35354220.2912730898
2.5894-2.64570.35544060.286736798
2.6457-2.70720.34643750.2804750099
2.7072-2.77490.33663840.2647729599
2.7749-2.84990.30673500.2322733999
2.8499-2.93380.29144060.2378737098
2.9338-3.02840.29913340.2457745298
3.0284-3.13660.35433700.2493733999
3.1366-3.26220.30953500.2279741698
3.2622-3.41050.29574490.2111729699
3.4105-3.59020.30473930.19637469100
3.5902-3.8150.24834080.17947408100
3.815-4.10930.23064000.1535739499
4.1093-4.52230.20233890.1417735198
4.5223-5.17550.16993510.1221742999
5.1755-6.51580.22553950.15357455100
6.5158-39.5740.20253600.1621738799
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)
10.5656-0.2689-0.08980.9677-0.07325.2546-0.02910.15580.0003-0.1022-0.0704-0.04870.2275-0.02350.08540.3907-0.0448-0.02920.451-0.00330.2806-11.35227.489-40.72
20.8366-0.23160.02131.1213-0.27143.14030.0083-0.0311-0.0575-0.0828-0.04740.0726-0.0252-0.24760.03490.247-0.0544-0.04630.3013-0.05380.2577-12.54426.87-3.493
30.84880.2078-0.08821.0669-0.35212.92090.0017-0.00160.08520.0927-0.0340.05630.0129-0.25990.02690.24440.04670.03450.2932-0.05320.2459-38.378-24.587-78.858
40.58780.12150.0820.9431-0.07375.2064-0.01-0.18150.02450.1191-0.0792-0.0609-0.149-0.00270.0670.37760.04040.01330.4365-0.00590.2805-37.276-25.291-41.528
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 302:722 )A302 - 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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