SASDEJ3: Truncated neutophil cytosol factor 1, p47phox [1-342]

基本情報

• 登録情報: データベース: SASBDB / ID: SASDEJ3

試料

Truncated neutophil cytosol factor 1, p47phox [1-342]

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

機能・相同性

分子機能:

reactive oxygen species biosynthetic process / regulation of respiratory burst involved in inflammatory response / superoxide-generating NADPH oxidase activator activity / phagolysosome / superoxide-generating NAD(P)H oxidase activity / positive regulation of epidermal growth factor-activated receptor 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 / cytoplasmic side of plasma membrane / VEGFA-VEGFR2 Pathway / cellular response to reactive oxygen species / SH3 domain binding / 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 / cytoplasm / cytosol

ドメイン・相同性:

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

構成要素:

Neutrophil cytosol factor 1

• 生物種: Homo sapiens (ヒト)
• 引用: ジャーナル: J Biol Chem / 年: 2019; タイトル: Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.; 著者: Cornelia S Ziegler / Leïla Bouchab / Marc Tramier / Dominique Durand / Franck Fieschi / Sophie Dupré-Crochet / Fabienne Mérola / Oliver Nüße / Marie Erard / ; 要旨: 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.

登録者

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

モデル

• モデル #2308: ; タイプ: atomic / カイ2乗値: 0.971 / P-value: 0.419653; Omokage検索でこの集合体の類似形状データを探す (詳細)

試料

• 試料: 名称: Truncated neutophil cytosol factor 1, p47phox [1-342]; 試料濃度: 0.90-3.60
• バッファ: 名称: 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 5% glycerol; pH: 7.5

要素 #1261

名称: p47phox / タイプ: protein / 記述: Neutophil cytosol factor 1 / 分子量: 40.398 / 分子数: 1 / 由来: Homo sapiens / 参照: UniProt: P14598
配列:

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 PG

実験情報

• ビーム: 設備名称: IBBMC Bruker Nanostar / 地域: Orsay / 国: France / 線源: X-ray in house / 波長: 0.154 Å / スペクトロメータ・検出器間距離: 0.662 mm
• 検出器: 名称: VÅNTEC-2000 / タイプ: MikroGap / Pixsize x: 200 mm

スキャン

タイトル: Truncated neutophil cytosol factor 1, p47phox [1-342]
測定日: 2009年10月16日 / 保管温度: 6 °C / セル温度: 6 °C / 照射時間: 3600 sec. / フレーム数: 11 / 単位: 1/A /

	Min	Max
Q	0.0142	0.4521

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 240 /

	Min	Max
Q	0.014225	0.354087
P(R) point	1	240
R	0	100

結果

カーブのタイプ: extrapolated

	実験値	Standard	Standard error	Porod
分子量	39.4 kDa	38 kDa	4	38.5 kDa
体積	-	-	-	57.8 nm3

	P(R)	P(R) error	Guinier	Guinier error
前方散乱 I0	0.02836	0.0003	0.0287	0.0003
慣性半径, Rg	2.63 nm	0.06	2.64 nm	0.06

	Min	Max	Error
D	-	10	0.5
Guinier point	1	23	-