SASDEL3: The neutrophil cytosol factor 2 (p67phox) subunit of phagocyte NA...

Basic information

• Entry: Database: SASBDB / ID: SASDEL3

Sample

The neutrophil cytosol factor 2 (p67phox) subunit of phagocyte NADPH oxidase

• Neutrophil cytosol factor 2 (protein), p67phox, Homo sapiens

Function / homology

Function:

superoxide-generating NADPH oxidase activator activity / phagolysosome / superoxide-generating NAD(P)H oxidase activity / Cross-presentation of particulate exogenous antigens (phagosomes) / NADPH oxidase complex / respiratory burst / ROS and RNS production in phagocytes / superoxide anion generation / superoxide metabolic process / Detoxification of Reactive Oxygen Species / RHO GTPases Activate NADPH Oxidases / RAC2 GTPase cycle / RAC3 GTPase cycle / cellular defense response / phagocytosis / RAC1 GTPase cycle / acrosomal vesicle / small GTPase binding / VEGFA-VEGFR2 Pathway / electron transfer activity / innate immune response / membrane / plasma membrane / cytosol

Domain/homology:

Neutrophil cytosol factor 2, PB1 domain / Neutrophil cytosol factor 2 / Neutrophil cytosol factor 2, SH3 domain 1 / PB1 domain / PB1 domain / PB1 domain profile. / PB1 domain / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / SH3 domain / Src homology 3 domains / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Tetratricopeptide-like helical domain superfamily

Component:

Neutrophil cytosol factor 2

• 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 #2310: ; Type: atomic / Chi-square value: 1.304 / P-value: 0.000001; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: The neutrophil cytosol factor 2 (p67phox) subunit of phagocyte NADPH oxidase; Specimen concentration: 0.30-3.80
• Buffer: Name: 20 mM HEPES, 50 mM NaCl, 1 mM EDTA, 2 mM DTT, 5% glycerol; pH: 8

Entity #1263

Name: p67phox / Type: protein / Description: Neutrophil cytosol factor 2 / Formula weight: 60.666 / Num. of mol.: 1 / Source: Homo sapiens / References: UniProt: P19878
Sequence:

GPLGSPNSAR MSLVEAISLW NEGVLAADKK DWKGALDAFS AVQDPHSRIC FNIGCMYTIL KNMTEAEKAF TRSINRDKHL AVAYFQRGML YYQTEKYDLA IKDLKEALIQ LRGNQLIDYK ILGLQFKLFA CEVLYNIAFM YAKKEEWKKA EEQLALATSM KSEPRHSKID KAMECVWKQK LYEPVVIPVG KLFRPNERQV AQLAKKDYLG KATVVASVVD QDSFSGFAPL QPQAAEPPPR PKTPEIFRAL EGEAHRVLFG FVPETKEELQ VMPGNIVFVL KKGNDNWATV MFNGQKGLVP CNYLEPVELR IHPQQQPQEE SSPQSDIPAP PSSKAPGRPQ LSPGQKQKEE PKEVKLSVPM PYTLKVHYKY TVVMKTQPGL PYSQVRDMVS KKLELRLEHT KLSYRPRDSN ELVPLSEDSM KDAWGQVKNY CLTLWCENTV GDQGFPDEPK ESEKADANNQ TTEPQLKKGS QVEALFSYEA TQPEDLEFQE GDIILVLSKV NEEWLEGESK GKVGIFPKVF VEDSATTDLE STRREV

Experimental information

• Beam: Instrument name: LURE D24 / City: Orsay / 国: France / Type of source: X-ray synchrotron / Wavelength: 0.1488 Å / Dist. spec. to detc.: 1.378 mm
• Detector: Name: Custom / Type: Linear gas detector / Pixsize x: 605 mm

Scan

Title: The neutrophil cytosol factor 2 (p67phox) subunit of the phagocyte NADPH oxidase
Measurement date: Apr 9, 2003 / Storage temperature: 4 °C / Cell temperature: 4 °C / Exposure time: 200 sec. / Number of frames: 8 / Unit: 1/A /

	Min	Max
Q	0.0148	0.3374

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 156 /

	Min	Max
Q	0.014831	0.30218
P(R) point	1	156
R	0	160

Result

Type of curve: extrapolated

	Experimental	Standard	Standard error	Porod
MW	62.4 kDa	60.4 kDa	6	75.3 kDa
Volume	-	-	-	113 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	0.05088	0.0006	0.05	0.0006
Radius of gyration, Rg	4.54 nm	0.1	4.27 nm	0.1

	Min	Max	Error
D	-	16	1
Guinier point	1	8	-