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- PDB-6nhf: Structure of human endothelial nitric oxide synthase heme domain ... -

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Basic information

Entry
Database: PDB / ID: 6nhf
TitleStructure of human endothelial nitric oxide synthase heme domain in complex with (S)-6-(2,3-difluoro-5-(2-(1-methylazetidin-2-yl)ethyl)phenethyl)-4-methylpyridin-2-amine
ComponentsEndothelial nitric oxide synthase splice variant eNOS13A
KeywordsOXIDOREDUCTASE/OXIDOREDUCTASE inhibitor / nitric oxide synthase inhibitor complex heme enzyme / OXIDOREDUCTASE / OXIDOREDUCTASE-OXIDOREDUCTASE inhibitor complex
Function / homology
Function and homology information


regulation of the force of heart contraction by chemical signal / NOSIP mediated eNOS trafficking / negative regulation of muscle hyperplasia / tetrahydrobiopterin metabolic process / NOSTRIN mediated eNOS trafficking / smooth muscle hyperplasia / regulation of nervous system process / superoxide-generating NAD(P)H oxidase activity / ovulation from ovarian follicle / pulmonary valve morphogenesis ...regulation of the force of heart contraction by chemical signal / NOSIP mediated eNOS trafficking / negative regulation of muscle hyperplasia / tetrahydrobiopterin metabolic process / NOSTRIN mediated eNOS trafficking / smooth muscle hyperplasia / regulation of nervous system process / superoxide-generating NAD(P)H oxidase activity / ovulation from ovarian follicle / pulmonary valve morphogenesis / response to fluid shear stress / negative regulation of biomineral tissue development / Nitric oxide stimulates guanylate cyclase / regulation of systemic arterial blood pressure by endothelin / ROS and RNS production in phagocytes / tetrahydrobiopterin binding / arginine binding / aortic valve morphogenesis / endocardial cushion morphogenesis / ventricular septum morphogenesis / positive regulation of Notch signaling pathway / cadmium ion binding / negative regulation of calcium ion transport / negative regulation of potassium ion transport / negative regulation of platelet activation / actin monomer binding / nitric oxide mediated signal transduction / nitric-oxide synthase (NADPH) / blood vessel remodeling / positive regulation of blood vessel endothelial cell migration / nitric-oxide synthase activity / endothelial cell migration / L-arginine catabolic process / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / regulation of sodium ion transport / negative regulation of blood pressure / response to hormone / nitric oxide metabolic process / eNOS activation / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / nitric oxide biosynthetic process / homeostasis of number of cells within a tissue / removal of superoxide radicals / lung development / cell redox homeostasis / lipopolysaccharide-mediated signaling pathway / blood vessel diameter maintenance / VEGFR2 mediated vascular permeability / mitochondrion organization / negative regulation of smooth muscle cell proliferation / establishment of localization in cell / caveola / potassium ion transport / regulation of blood pressure / vasodilation / positive regulation of angiogenesis / endocytic vesicle membrane / calcium ion transport / FMN binding / NADP binding / flavin adenine dinucleotide binding / response to heat / scaffold protein binding / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / angiogenesis / response to lipopolysaccharide / in utero embryonic development / cytoskeleton / calmodulin binding / Extra-nuclear estrogen signaling / Golgi membrane / negative regulation of cell population proliferation / heme binding / positive regulation of gene expression / Golgi apparatus / metal ion binding / nucleus / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Bovine Endothelial Nitric Oxide Synthase Heme Domain; Chain: A,domain 3 / Nitric Oxide Synthase; Chain A, domain 3 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase;Heme Domain; Chain A, domain 2 / Nitric Oxide Synthase;Heme Domain;Chain A domain 2 / Nitric-oxide synthase, eukaryote / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, domain 2 superfamily ...Bovine Endothelial Nitric Oxide Synthase Heme Domain; Chain: A,domain 3 / Nitric Oxide Synthase; Chain A, domain 3 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase; Chain A, domain 1 / Nitric Oxide Synthase;Heme Domain; Chain A, domain 2 / Nitric Oxide Synthase;Heme Domain;Chain A domain 2 / 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 / Flavoprotein-like superfamily / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
GADOLINIUM ATOM / 5,6,7,8-TETRAHYDROBIOPTERIN / PROTOPORPHYRIN IX CONTAINING FE / Chem-KNV / Nitric oxide synthase 3 / Nitric oxide synthase 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.83 Å
AuthorsChreifi, G. / Li, H. / Poulos, T.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM57353 United States
CitationJournal: J. Med. Chem. / Year: 2019
Title: Optimization of Blood-Brain Barrier Permeability with Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibitors Having a 2-Aminopyridine Scaffold.
Authors: Do, H.T. / Li, H. / Chreifi, G. / Poulos, T.L. / Silverman, R.B.
History
DepositionDec 21, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr1_symmetry / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Endothelial nitric oxide synthase splice variant eNOS13A
B: Endothelial nitric oxide synthase splice variant eNOS13A
C: Endothelial nitric oxide synthase splice variant eNOS13A
D: Endothelial nitric oxide synthase splice variant eNOS13A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,58339
Polymers197,3834
Non-polymers8,20035
Water10,863603
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A: Endothelial nitric oxide synthase splice variant eNOS13A
B: Endothelial nitric oxide synthase splice variant eNOS13A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,89620
Polymers98,6922
Non-polymers4,20518
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11240 Å2
ΔGint-151 kcal/mol
Surface area34300 Å2
MethodPISA
2
C: Endothelial nitric oxide synthase splice variant eNOS13A
D: Endothelial nitric oxide synthase splice variant eNOS13A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,68719
Polymers98,6922
Non-polymers3,99517
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10870 Å2
ΔGint-154 kcal/mol
Surface area33220 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.336, 152.557, 108.524
Angle α, β, γ (deg.)90.00, 90.70, 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
Endothelial nitric oxide synthase splice variant eNOS13A


Mass: 49345.770 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: endothelial / Plasmid: pCWori / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0S0A6, UniProt: P29474*PLUS

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Non-polymers , 9 types, 638 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
#4: Chemical
ChemComp-KNV / 6-[2-(2,3-difluoro-5-{2-[(2S)-1-methylazetidin-2-yl]ethyl}phenyl)ethyl]-4-methylpyridin-2-amine


Mass: 345.429 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C20H25F2N3
#5: Chemical
ChemComp-BTB / 2-[BIS-(2-HYDROXY-ETHYL)-AMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / BIS-TRIS BUFFER


Mass: 209.240 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C8H19NO5 / Comment: pH buffer*YM
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#8: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
#9: Chemical
ChemComp-GD / GADOLINIUM ATOM


Mass: 157.250 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Gd
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 603 / Source method: isolated from a natural source / Formula: H2O

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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: 2.69 Å3/Da / Density % sol: 54.3 % / Description: rods
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 12-15% PEG3350, 0.1M BIS-TRIS 0.2-0.3M MG ACETATE, 0.1M GdCl3 10% glycerol, 5 mM TCEP

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.2 / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 300K / Detector: PIXEL / Date: Nov 8, 2018 / Details: mirrors
RadiationMonochromator: graphite / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.83→60 Å / Num. obs: 168956 / % possible obs: 99.8 % / Observed criterion σ(I): -3 / Redundancy: 7.5 % / CC1/2: 0.998 / Rmerge(I) obs: 0.115 / Rpim(I) all: 0.045 / Rsym value: 0.115 / Net I/σ(I): 8.7
Reflection shellResolution: 1.83→1.88 Å / Redundancy: 7.4 % / Rmerge(I) obs: 2.321 / Mean I/σ(I) obs: 0.8 / Num. unique obs: 8377 / CC1/2: 0.35 / Rpim(I) all: 0.909 / Rsym value: 2.321 / % possible all: 99.2

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Processing

Software
NameVersionClassification
PHENIX1.11.1-2575_1496refinement
XDSdata reduction
Aimlessdata scaling
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 4DIP

4dip


Resolution: 1.83→49.497 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 0.06 / Phase error: 27.09
RfactorNum. reflection% reflectionSelection details
Rfree0.2187 16821 5.02 %random
Rwork0.1828 ---
obs0.1846 168894 99.73 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.83→49.497 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12824 0 516 603 13943
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00813812
X-RAY DIFFRACTIONf_angle_d1.11918828
X-RAY DIFFRACTIONf_dihedral_angle_d14.8518065
X-RAY DIFFRACTIONf_chiral_restr0.0551957
X-RAY DIFFRACTIONf_plane_restr0.0052414
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.83-1.85080.3835760.353710614X-RAY DIFFRACTION99
1.8508-1.87260.34275910.339110556X-RAY DIFFRACTION100
1.8726-1.89540.38245380.339710573X-RAY DIFFRACTION100
1.8954-1.91940.35234930.326110687X-RAY DIFFRACTION99
1.9194-1.94470.35315200.317410552X-RAY DIFFRACTION100
1.9447-1.97130.33495980.299210650X-RAY DIFFRACTION100
1.9713-1.99950.34815230.289810480X-RAY DIFFRACTION100
1.9995-2.02930.29145810.280310627X-RAY DIFFRACTION100
2.0293-2.0610.31376290.276710523X-RAY DIFFRACTION100
2.061-2.09480.33356050.274910544X-RAY DIFFRACTION100
2.0948-2.13090.28765980.253610606X-RAY DIFFRACTION100
2.1309-2.16970.28015540.228210520X-RAY DIFFRACTION100
2.1697-2.21140.25145310.225110748X-RAY DIFFRACTION100
2.2114-2.25660.26545630.224710561X-RAY DIFFRACTION100
2.2566-2.30560.23945600.208810647X-RAY DIFFRACTION100
2.3056-2.35930.25175700.207310580X-RAY DIFFRACTION100
2.3593-2.41820.24975480.196110630X-RAY DIFFRACTION100
2.4182-2.48360.24226110.19410612X-RAY DIFFRACTION100
2.4836-2.55670.22195250.188410613X-RAY DIFFRACTION100
2.5567-2.63920.21756050.175710572X-RAY DIFFRACTION100
2.6392-2.73360.21715970.177510635X-RAY DIFFRACTION100
2.7336-2.8430.23395420.184110629X-RAY DIFFRACTION100
2.843-2.97240.23415620.184510674X-RAY DIFFRACTION100
2.9724-3.1290.21175050.174510660X-RAY DIFFRACTION100
3.129-3.32510.21575550.172710612X-RAY DIFFRACTION100
3.3251-3.58170.20015770.159510533X-RAY DIFFRACTION100
3.5817-3.9420.17725450.147910635X-RAY DIFFRACTION100
3.942-4.51210.16975650.134910563X-RAY DIFFRACTION99
4.5121-5.68350.17655530.139510591X-RAY DIFFRACTION100
5.6835-49.51520.1825010.174410625X-RAY DIFFRACTION99
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)
10.96590.2125-0.28032.308-0.34581.95080.21990.38890.37690.00680.11640.3887-0.5817-0.5485-0.18360.46810.1790.15650.55360.22410.465563.781231.7497-185.136
21.2331-0.2398-0.40471.3635-0.51662.49430.0533-0.04130.08210.25070.0705-0.0056-0.0924-0.0982-0.11320.2355-0.0426-0.00840.19530.00720.242873.43978.6117-157.2342
31.0939-0.24560.58932.0119-0.34722.17710.1946-0.3294-0.37830.04620.09360.35680.5656-0.3495-0.15740.4332-0.0509-0.07230.36140.14490.422792.5285-34.1836-194.889
41.01790.17910.39880.7623-0.09612.1235-0.01990.0542-0.0128-0.1040.0411-0.0249-0.09270.1546-0.02690.28930.0493-0.01030.2225-0.00640.263102.4282-10.2787-222.1138
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 68:480 )A68 - 480
2X-RAY DIFFRACTION2( CHAIN B AND RESID 67:480 )B67 - 480
3X-RAY DIFFRACTION3( CHAIN C AND RESID 68:480 )C68 - 480
4X-RAY DIFFRACTION4( CHAIN D AND RESID 68:480 )D68 - 480

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