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- EMDB-40970: DSBU crosslinked nNOS-CaM oxygenase homodimer -

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Basic information

Entry
Database: EMDB / ID: EMD-40970
TitleDSBU crosslinked nNOS-CaM oxygenase homodimer
Map datanNOS-CaM-DSBU map
Sample
  • Complex: DSBU crosslinked CaM-bound nNOS homodimer
    • Protein or peptide: Nitric oxide synthase 1
  • Ligand: ZINC ION
  • Ligand: ARGININE
  • Ligand: 5,6,7,8-TETRAHYDROBIOPTERIN
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
KeywordsComplex / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


Nitric oxide stimulates guanylate cyclase / negative regulation of hepatic stellate cell contraction / positive regulation of adenylate cyclase-activating adrenergic receptor signaling pathway / retrograde trans-synaptic signaling by nitric oxide / synaptic signaling by nitric oxide / negative regulation of iron ion transmembrane transport / ROS and RNS production in phagocytes / azurophil granule / positive regulation of sodium ion transmembrane transport / negative regulation of vasoconstriction ...Nitric oxide stimulates guanylate cyclase / negative regulation of hepatic stellate cell contraction / positive regulation of adenylate cyclase-activating adrenergic receptor signaling pathway / retrograde trans-synaptic signaling by nitric oxide / synaptic signaling by nitric oxide / negative regulation of iron ion transmembrane transport / ROS and RNS production in phagocytes / azurophil granule / positive regulation of sodium ion transmembrane transport / negative regulation of vasoconstriction / postsynaptic specialization, intracellular component / nitric oxide metabolic process / positive regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / response to nitric oxide / Ion homeostasis / negative regulation of cytosolic calcium ion concentration / 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 / behavioral response to cocaine / calyx of Held / regulation of neurogenesis / negative regulation of serotonin uptake / nitric-oxide synthase (NADPH) / regulation of postsynaptic membrane potential / response to vitamin E / sodium channel regulator activity / nitric oxide mediated signal transduction / postsynaptic density, intracellular component / nitric-oxide synthase activity / negative regulation of insulin secretion / xenobiotic catabolic process / multicellular organismal response to stress / arginine catabolic process / NADPH binding / striated muscle contraction / regulation of sodium ion transport / nitric oxide-cGMP-mediated signaling / nitric oxide biosynthetic process / T-tubule / cellular response to epinephrine stimulus / sarcoplasmic reticulum membrane / negative regulation of blood pressure / photoreceptor inner segment / response to hormone / response to nutrient levels / secretory granule / sarcoplasmic reticulum / positive regulation of long-term synaptic potentiation / cell periphery / response to activity / female pregnancy / establishment of localization in cell / phosphoprotein binding / response to nicotine / response to lead ion / establishment of protein localization / potassium ion transport / caveola / sarcolemma / response to organic cyclic compound / cellular response to growth factor stimulus / response to peptide hormone / Z disc / response to estrogen / cellular response to mechanical stimulus / vasodilation / calcium-dependent protein binding / calcium ion transport / FMN binding / positive regulation of peptidyl-serine phosphorylation / flavin adenine dinucleotide binding / NADP binding / ATPase binding / response to heat / scaffold protein binding / nuclear membrane / response to ethanol / negative regulation of neuron apoptotic process / transmembrane transporter binding / mitochondrial outer membrane / response to lipopolysaccharide / dendritic spine / postsynaptic density / cytoskeleton / calmodulin binding / response to hypoxia / membrane raft / negative regulation of cell population proliferation / glutamatergic synapse / dendrite / heme binding / synapse / negative regulation of apoptotic process / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / enzyme binding / positive regulation of transcription by RNA polymerase II
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
Nitric oxide synthase 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsLee K / Pospiech TH / Southworth D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM077430 United States
CitationJournal: J Biol Chem / Year: 2024
Title: Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.
Authors: Dana Felker / Kanghyun Lee / Thomas H Pospiech / Yoshihiro Morishima / Haoming Zhang / Miranda Lau / Daniel R Southworth / Yoichi Osawa /
Abstract: Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The ...Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.
History
DepositionJun 2, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40970.png

Downloads & links

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Map

FileDownload / File: emd_40970.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationnNOS-CaM-DSBU map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 340 pix.
= 310.08 Å		0.91 Å/pix.
x 340 pix.
= 310.08 Å		0.91 Å/pix.
x 340 pix.
= 310.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.912 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.8
Minimum - Maximum-6.7172313 - 8.752471999999999
Average (Standard dev.)-0.00063410023 (±0.1412107)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 310.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: nNOS-CaM-DSBU half map A

Fileemd_40970_half_map_1.map
AnnotationnNOS-CaM-DSBU half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: nNOS-CaM-DSBU half map B

Fileemd_40970_half_map_2.map
AnnotationnNOS-CaM-DSBU half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DSBU crosslinked CaM-bound nNOS homodimer

EntireName: DSBU crosslinked CaM-bound nNOS homodimer
Components
  • Complex: DSBU crosslinked CaM-bound nNOS homodimer
    • Protein or peptide: Nitric oxide synthase 1
  • Ligand: ZINC ION
  • Ligand: ARGININE
  • Ligand: 5,6,7,8-TETRAHYDROBIOPTERIN
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE

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Supramolecule #1: DSBU crosslinked CaM-bound nNOS homodimer

SupramoleculeName: DSBU crosslinked CaM-bound nNOS homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Nitric oxide synthase 1

MacromoleculeName: Nitric oxide synthase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitric-oxide synthase (NADPH)
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 160.769562 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEENTFGVQQ IQPNVISVRL FKRKVGGLGF LVKERVSKPP VIISDLIRGG AAEQSGLIQA GDIILAVNDR PLVDLSYDSA LEVLRGIAS ETHVVLILRG PEGFTTHLET TFTGDGTPKT IRVTQPLGPP TKAVDLSHQP SASKDQSLAV DRVTGLGNGP Q HAQGHGQG ...String:
MEENTFGVQQ IQPNVISVRL FKRKVGGLGF LVKERVSKPP VIISDLIRGG AAEQSGLIQA GDIILAVNDR PLVDLSYDSA LEVLRGIAS ETHVVLILRG PEGFTTHLET TFTGDGTPKT IRVTQPLGPP TKAVDLSHQP SASKDQSLAV DRVTGLGNGP Q HAQGHGQG AGSVSQANGV AIDPTMKSTK ANLQDIGEHD ELLKEIEPVL SILNSGSKAT NRGGPAKAEM KDTGIQVDRD LD GKSHKAP PLGGDNDRVF NDLWGKDNVP VILNNPYSEK EQSPTSGKQS PTKNGSPSRC PRFLKVKNWE TDVVLTDTLH LKS TLETGC TEHICMGSIM LPSQHTRKPE DVRTKDQLFP LAKEFLDQYY SSIKRFGSKA HMDRLEEVNK EIESTSTYQL KDTE LIYGA KHAWRNASRC VGRIQWSKLQ VFDARDCTTA HGMFNYICNH VKYATNKGNL RSAITIFPQR TDGKHDFRVW NSQLI RYAG YKQPDGSTLG DPANVQFTEI CIQQGWKAPR GRFDVLPLLL QANGNDPELF QIPPELVLEV PIRHPKFDWF KDLGLK WYG LPAVSNMLLE IGGLEFSACP FSGWYMGTEI GVRDYCDNSR YNILEEVAKK MDLDMRKTSS LWKDQALVEI NIAVLYS FQ SDKVTIVDHH SATESFIKHM ENEYRCRGGC PADWVWIVPP MSGSITPVFH QEMLNYRLTP SFEYQPDPWN THVWKGTN G TPTKRRAIGF KKLAEAVKFS AKLMGQAMAK RVKATILYAT ETGKSQAYAK TLCEIFKHAF DAKAMSMEEY DIVHLEHEA LVLVVTSTFG NGDPPENGEK FGCALMEMRH PNSVQEERKS YKVRFNSVSS YSDSRKSSGD GPDLRDNFES TGPLANVRFS VFGLGSRAY PHFCAFGHAV DTLLEELGGE RILKMREGDE LCGQEEAFRT WAKKVFKAAC DVFCVGDDVN IEKPNNSLIS N DRSWKRNK FRLTYVAEAP DLTQGLSNVH KKRVSAARLL SRQNLQSPKF SRSTIFVRLH TNGNQELQYQ PGDHLGVFPG NH EDLVNAL IERLEDAPPA NHVVKVEMLE ERNTALGVIS NWKDESRLPP CTIFQAFKYY LDITTPPTPL QLQQFASLAT NEK EKQRLL VLSKGLQEYE EWKWGKNPTM VEVLEEFPSI QMPATLLLTQ LSLLQPRYYS ISSSPDMYPD EVHLTVAIVS YHTR DGEGP VHHGVCSSWL NRIQADDVVP CFVRGAPSFH LPRNPQVPCI LVGPGTGIAP FRSFWQQRQF DIQHKGMNPC PMVLV FGCR QSKIDHIYRE ETLQAKNKGV FRELYTAYSR EPDRPKKYVQ DVLQEQLAES VYRALKEQGG HIYVCGDVTM AADVLK AIQ RIMTQQGKLS EEDAGVFISR LRDDNRYHED IFGVTLRTYE VTNRLRSESI AFIEESKKDA DEVFSS

UniProtKB: Nitric oxide synthase 1

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: ARGININE

MacromoleculeName: ARGININE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ARG
Molecular weightTheoretical: 175.209 Da
Chemical component information

ChemComp-ARG:
ARGININE

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Macromolecule #4: 5,6,7,8-TETRAHYDROBIOPTERIN

MacromoleculeName: 5,6,7,8-TETRAHYDROBIOPTERIN / type: ligand / ID: 4 / Number of copies: 2 / Formula: H4B
Molecular weightTheoretical: 241.247 Da
Chemical component information

ChemComp-H4B:
5,6,7,8-TETRAHYDROBIOPTERIN / neurotransmitter*YM

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Macromolecule #5: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 5 / Number of copies: 2 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 294924
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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