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- PDB-6cic: Structure of the human nitric oxide synthase R354A/G357D mutant h... -

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Basic information

Entry
Database: PDB / ID: 6cic
TitleStructure of the human nitric oxide synthase R354A/G357D mutant heme domain in complex with N-(1-(2-(Ethyl(methyl)amino)ethyl)-1,2,3,4-tetrahydroquino-lin-6-yl)thiophene-2-carboximidamide
ComponentsNitric oxide synthase, brain
KeywordsOXIDOREDUCTASE / nitric oxide synthase inhibitor complex heme enzyme
Function / homology
Function and homology information


positive regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / 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 adenylate cyclase-activating G protein-coupled receptor signaling pathway / 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 / 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
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, domain 2 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 3 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, 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 / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
Chem-7R2 / 5,6,7,8-TETRAHYDROBIOPTERIN / PROTOPORPHYRIN IX CONTAINING FE / Nitric oxide synthase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.752 Å
AuthorsLi, H. / Poulos, T.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM57353 United States
CitationJournal: Biochemistry / Year: 2018
Title: Structural Basis for Isoform Selective Nitric Oxide Synthase Inhibition by Thiophene-2-carboximidamides.
Authors: Li, H. / Evenson, R.J. / Chreifi, G. / Silverman, R.B. / Poulos, T.L.
History
DepositionFeb 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 31, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 7, 2018Group: Data collection / Database references / Category: citation / citation_author / Item: _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 21, 2018Group: 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 1.3Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Oct 4, 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 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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.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

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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)100,21911
Polymers97,5692
Non-polymers2,6509
Water13,583754
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9820 Å2
ΔGint-86 kcal/mol
Surface area33770 Å2
MethodPISA
Unit cell
Length a, b, c (Å)52.318, 122.157, 165.131
Angle α, β, γ (deg.)90.00, 90.00, 90.00
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 (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P29475, nitric-oxide synthase (NADPH)

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Non-polymers , 6 types, 763 molecules

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


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


Mass: 241.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H15N5O3 / Comment: neurotransmitter*YM
#4: Chemical ChemComp-7R2 / N-(1-{2-[ethyl(methyl)amino]ethyl}-1,2,3,4-tetrahydroquinolin-6-yl)thiophene-2-carboximidamide


Mass: 342.502 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H26N4S
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / 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 / Water


Mass: 18.015 Da / Num. of mol.: 754 / 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: 2.74 Å3/Da / Density % sol: 55.2 % / Description: plate
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: 1.0332 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 18, 2017 / Details: mirrors
RadiationMonochromator: graphite / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 1.75→60 Å / Num. obs: 106791 / % possible obs: 99.6 % / Observed criterion σ(I): -3 / Redundancy: 8.8 % / CC1/2: 0.995 / Rmerge(I) obs: 0.121 / Rpim(I) all: 0.043 / Rsym value: 0.121 / Net I/σ(I): 8.2
Reflection shellResolution: 1.75→1.8 Å / Redundancy: 7.9 % / Rmerge(I) obs: 5.054 / Mean I/σ(I) obs: 0.4 / Num. unique obs: 4852 / CC1/2: 0.286 / Rpim(I) all: 1.864 / Rsym value: 5.054 / % possible all: 93.3

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Processing

Software
NameVersionClassification
PHENIX(1.11.1-2575_1496: ???)refinement
XDSdata reduction
Aimlessdata scaling
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 4UH5

4uh5


Resolution: 1.752→37.921 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 28.18
RfactorNum. reflection% reflectionSelection details
Rfree0.2039 9735 4.88 %random
Rwork0.172 ---
obs0.1735 103944 96.98 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.752→37.921 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6853 0 181 754 7788
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0067284
X-RAY DIFFRACTIONf_angle_d0.9939916
X-RAY DIFFRACTIONf_dihedral_angle_d17.3674269
X-RAY DIFFRACTIONf_chiral_restr0.0521020
X-RAY DIFFRACTIONf_plane_restr0.0051260
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7517-1.77160.43352480.3895508X-RAY DIFFRACTION84
1.7716-1.79240.3883120.36026101X-RAY DIFFRACTION94
1.7924-1.81430.38522800.34456126X-RAY DIFFRACTION93
1.8143-1.83720.34793310.3336113X-RAY DIFFRACTION95
1.8372-1.86140.38773210.33916202X-RAY DIFFRACTION95
1.8614-1.88690.33753530.33636156X-RAY DIFFRACTION95
1.8869-1.91390.37733310.32426207X-RAY DIFFRACTION95
1.9139-1.94240.35492710.30516287X-RAY DIFFRACTION95
1.9424-1.97280.30743220.28746113X-RAY DIFFRACTION95
1.9728-2.00510.28983830.27326224X-RAY DIFFRACTION96
2.0051-2.03970.30442970.25796262X-RAY DIFFRACTION96
2.0397-2.07680.29723360.24616338X-RAY DIFFRACTION97
2.0768-2.11670.30013380.23486349X-RAY DIFFRACTION98
2.1167-2.15990.25433330.22266314X-RAY DIFFRACTION98
2.1599-2.20690.25813180.21386384X-RAY DIFFRACTION98
2.2069-2.25820.21593200.20366457X-RAY DIFFRACTION98
2.2582-2.31470.25043030.19956357X-RAY DIFFRACTION98
2.3147-2.37720.23493570.19556484X-RAY DIFFRACTION98
2.3772-2.44720.19893470.18736293X-RAY DIFFRACTION98
2.4472-2.52620.20623660.17786394X-RAY DIFFRACTION99
2.5262-2.61640.22773180.1776521X-RAY DIFFRACTION99
2.6164-2.72120.22513670.1776413X-RAY DIFFRACTION99
2.7212-2.8450.20172850.17656509X-RAY DIFFRACTION99
2.845-2.99490.19073260.1616468X-RAY DIFFRACTION99
2.9949-3.18250.18723070.16216498X-RAY DIFFRACTION99
3.1825-3.4280.1863410.14586535X-RAY DIFFRACTION100
3.428-3.77270.16723590.13226478X-RAY DIFFRACTION100
3.7727-4.3180.16093390.12576520X-RAY DIFFRACTION100
4.318-5.43760.1533300.12376481X-RAY DIFFRACTION100
5.4376-37.93030.16462960.14786552X-RAY DIFFRACTION100
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)
10.8312-0.16510.0610.86660.27942.06840.0002-0.04190.0363-0.0225-0.0412-0.0380.03430.12620.03830.1653-0.02580.030.22170.02850.211117.4141249.6593361.1207
20.7683-0.144-0.12110.85130.0363.26220.04940.10380.0317-0.0845-0.05890.0734-0.0845-0.17330.00520.25660.01480.02950.3195-0.02080.2811115.6837248.4107323.7108
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resid 302:722)
2X-RAY DIFFRACTION2(chain B and resid 304:722)

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