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

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Basic information

Entry
Database: PDB / ID: 8fgf
TitleStructure of human neuronal nitric oxide synthase R354A/G357D mutant heme domain in complex with 6-(5-(2-(dimethylamino)ethyl)-2,3-difluorophenethyl)pyridin-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) / multicellular organismal response to stress / regulation of ryanodine-sensitive calcium-release channel activity / sodium channel regulator activity / nitric oxide mediated signal transduction / nitric-oxide synthase activity / xenobiotic catabolic process / 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 / cellular response to growth factor stimulus / sarcolemma / 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-XVO / Nitric oxide synthase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.83 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,23712
Polymers97,5692
Non-polymers2,66810
Water12,935718
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10060 Å2
ΔGint-85 kcal/mol
Surface area34340 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.397, 121.940, 164.895
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AB

#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.: 2 / 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, 728 molecules

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


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-H4B / 5,6,7,8-TETRAHYDROBIOPTERIN


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


Mass: 305.366 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21F2N3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 718 / 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 PILATUS 6M / Detector: PIXEL / Date: Dec 17, 2019 / Details: mirrors
RadiationMonochromator: DOUBLE CRYSTAL SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.83→38.63 Å / Num. obs: 94083 / % possible obs: 99.9 % / Redundancy: 10.5 % / Biso Wilson estimate: 29.55 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.198 / Rpim(I) all: 0.062 / Rrim(I) all: 0.208 / Net I/σ(I): 10.3 / Num. measured all: 984180 / Scaling rejects: 234
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
1.83-1.8663.9822760845950.3381.7564.3610.799.7
10.02-38.63110.04873876690.9990.0150.053798.3

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Processing

Software
NameVersionClassification
Aimless0.2.8data scaling
PHENIX1.11.1_2575refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 4UH5

4uh5


Resolution: 1.83→37.9336 Å / SU ML: 0.25 / Cross valid method: FREE R-VALUE / σ(F): 0.01 / Phase error: 26.7 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2127 8846 4.92 %random
Rwork0.1757 170816 --
obs0.1776 93881 99.79 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 129.49 Å2 / Biso mean: 43.2268 Å2 / Biso min: 17.26 Å2
Refinement stepCycle: final / Resolution: 1.83→37.9336 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6748 0 205 718 7671
Biso mean--40.76 49.28 -
Num. residues----828
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0067203
X-RAY DIFFRACTIONf_angle_d0.9539805
X-RAY DIFFRACTIONf_chiral_restr0.0521012
X-RAY DIFFRACTIONf_plane_restr0.0051236
X-RAY DIFFRACTIONf_dihedral_angle_d17.7284205
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.83-1.85080.3883080.33675664597299
1.8508-1.87260.36763070.315557726079100
1.8726-1.89540.34663260.318455515877100
1.8954-1.91940.3512870.309857035990100
1.9194-1.94470.39612410.314257525993100
1.9447-1.97130.29823060.296556095915100
1.9713-1.99950.30353480.284456395987100
1.9995-2.02930.35073140.271257156029100
2.0293-2.0610.30182510.256856775928100
2.061-2.09480.29973170.257157736090100
2.0948-2.13090.31853130.251256105923100
2.1309-2.16970.29972700.244957636033100
2.1697-2.21140.27782770.225956825959100
2.2114-2.25650.23142880.208956855973100
2.2565-2.30560.23762770.203557266003100
2.3056-2.35920.25062960.195956865982100
2.3592-2.41820.22663160.190656896005100
2.4182-2.48360.24073290.187456716000100
2.4836-2.55660.20123080.17756755983100
2.5566-2.63910.26562960.175457116007100
2.6391-2.73340.21793070.185456775984100
2.7334-2.84280.2172540.173757606014100
2.8428-2.97220.20152870.166456995986100
2.9722-3.12880.19072910.159656825973100
3.1288-3.32470.18672880.152257356023100
3.3247-3.58120.18872870.147657186005100
3.5812-3.94130.1553190.13356675986100
3.9413-4.51080.16472960.127356835979100
4.5108-5.68010.152840.132657286012100
5.6801-37.93360.20212580.15915714597299
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)
10.8015-0.1630.17940.87770.20261.85610.0132-0.00060.0198-0.0525-0.0354-0.05620.10360.15270.01590.1523-0.01270.03650.1950.02920.1964117.577249.119360.606
20.6604-0.2-0.05420.79880.24883.0360.05330.09140.0059-0.0518-0.03240.0704-0.0258-0.0946-0.01310.19620.01750.03560.2445-0.01870.2208116.054247.659323.191
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

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