SASDEK3: The neutrophil cytosol factor 1 (p47phox) subunit of phagocyte NA...

Basic information

• Entry: Database: SASBDB / ID: SASDEK3

Sample

The neutrophil cytosol factor 1 (p47phox) subunit of phagocyte NADPH oxidase

• Neutrophil cytosol factor 1 (protein), p47phox, Homo sapiens

Function / homology

Function:

regulation of respiratory burst involved in inflammatory response / superoxide-generating NADPH oxidase activator activity / reactive oxygen species biosynthetic process / positive regulation of epidermal growth factor-activated receptor activity / phagolysosome / superoxide-generating NAD(P)H oxidase activity / Cross-presentation of particulate exogenous antigens (phagosomes) / NADPH oxidase complex / respiratory burst / cellular response to testosterone stimulus / ROS and RNS production in phagocytes / phosphatidylinositol-3,4-bisphosphate binding / superoxide anion generation / protein targeting to membrane / positive regulation of p38MAPK cascade / superoxide metabolic process / Detoxification of Reactive Oxygen Species / RHO GTPases Activate NADPH Oxidases / RAC2 GTPase cycle / RAC3 GTPase cycle / cellular defense response / RAC1 GTPase cycle / cellular response to cadmium ion / phosphatidylinositol binding / cellular response to glucose stimulus / positive regulation of JNK cascade / VEGFA-VEGFR2 Pathway / SH3 domain binding / cytoplasmic side of plasma membrane / cellular response to reactive oxygen species / electron transfer activity / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / innate immune response / neuronal cell body / dendrite / positive regulation of DNA-templated transcription / membrane / plasma membrane / cytosol / cytoplasm

Domain/homology:

Neutrophil cytosol factor 1 / Neutrophil cytosol factor 1, C-terminal / Neutrophil cytosol factor 1, PBR/AIR / Neutrophil cytosol factor 1, PX domain / Neutrophil cytosol factor 1, first SH3 domain / Neutrophil cytosol factor 1, second SH3 domain / NADPH oxidase subunit p47Phox, C terminal domain / Neutrophil cytosol factor 1, PBR/AIR / : / PhoX homologous domain, present in p47phox and p40phox. / PX domain profile. / PX domain / Phox homology / PX domain superfamily / SH3 domain / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain

Component:

Neutrophil cytosol factor 1

• Biological species: Homo sapiens (human)
• Citation: Journal: J Biol Chem / Year: 2019; Title: Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.; Authors: Cornelia S Ziegler / Leïla Bouchab / Marc Tramier / Dominique Durand / Franck Fieschi / Sophie Dupré-Crochet / Fabienne Mérola / Oliver Nüße / Marie Erard / ; Abstract: Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22) and three cytosolic subunits (p40, p47, and p67) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

Contact author

• Dominique DURAND (I2BC-CNRS, Paris, France)

Models

• Model #2309: ; Type: atomic / Chi-square value: 1.245; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: The neutrophil cytosol factor 1 (p47phox) subunit of phagocyte NADPH oxidase; Specimen concentration: 1.00-4.00
• Buffer: Name: 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 5% glycerol; pH: 7.5

Entity #1262

Name: p47phox / Type: protein / Description: Neutrophil cytosol factor 1 / Formula weight: 46.364 / Num. of mol.: 1 / Source: Homo sapiens / References: UniProt: P14598
Sequence:

GPLGSPNSAR MGDTFIRHIA LLGFEKRFVP SQHYVYMFLV KWQDLSEKVV YRRFTEIYEF HKTLKEMFPI EAGAINPENR IIPHLPAPKW FDGQRAAENR QGTLTEYCST LMSLPTKISR CPHLLDFFKV RPDDLKLPTD NQTKKPETYL MPKDGKSTAT DITGPIILQT YRAIADYEKT SGSEMALSTG DVVEVVEKSE SGWWFCQMKA KRGWIPASFL EPLDSPDETE DPEPNYAGEP YVAIKAYTAV EGDEVSLLEG EAVEVIHKLL DGWWVIRKDD VTGYFPSMYL QKSGQDVSQA QRQIKRGAPP RRSSIRNAHS IHQRSRKRLS QDAYRRNSVR FLQQRRRQAR PGPQSPGSPL EEERQTQRSK PQPAVPPRPS ADLILNRCSE STKRKLASAV CGRIVTD

Experimental information

• Beam: Instrument name: SOLEIL SWING / City: Saint-Aubin / 国: France / Type of source: X-ray synchrotron / Wavelength: 0.1 Å / Dist. spec. to detc.: 1.783 mm
• Detector: Name: AVIEX PCCD170170 / Type: CCD

Scan

Title: Neutrophil cytosol factor 1 (p47phox) subunit of the phagocyte NADPH oxidase
Measurement date: Apr 23, 2008 / Storage temperature: 10 °C / Cell temperature: 10 °C / Exposure time: 2 sec. / Number of frames: 20 / Unit: 1/A /

	Min	Max
Q	0.0104	0.5004

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 804 /

	Min	Max
Q	0.012963	0.35016
P(R) point	1	804
R	0	125

Result

Type of curve: extrapolated

	Experimental	Standard	Standard error	Porod
MW	47.7 kDa	44.1 kDa	4	51.1 kDa
Volume	-	-	-	76.7 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	0.03307	0.0003	0.0327	0.0003
Radius of gyration, Rg	3.32 nm	0.03	3.17 nm	0.03

	Min	Max	Error
D	-	12.5	1
Guinier point	7	66	-