SASDDT9: NADPH oxidase (H2O2 producing and [F-actin] oxidizing) MICAL1 (mo...

Basic information

• Entry: Database: SASBDB / ID: SASDDT9

Sample

NADPH oxidase (H2O2 producing and [F-actin] oxidizing) MICAL1 (monomer) in complex with Ras-related protein Rab 8 (MICAL1-Rab8 complex)

• [F-actin]-monooxygenase MICAL1 (monomer) (protein), Mical1, Homo sapiens
• Ras-related protein 8 (protein), Rab8

Function / homology

Function:

establishment of protein localization to endoplasmic reticulum membrane / endoplasmic reticulum tubular network organization / protein localization to basolateral plasma membrane / establishment of neuroblast polarity / hippocampal mossy fiber expansion / NADPH oxidase H202-forming activity / F-actin monooxygenase / NAD(P)H oxidase (H2O2-forming) / sulfur oxidation / endoplasmic reticulum tubular network / regulation of regulated secretory pathway / insulin-responsive compartment / regulated exocytosis / NAD(P)H oxidase H2O2-forming activity / polarized epithelial cell differentiation / oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen / exocytic vesicle / Golgi to plasma membrane protein transport / cadherin binding involved in cell-cell adhesion / Golgi to plasma membrane transport / RAB geranylgeranylation / myosin V binding / RAB GEFs exchange GTP for GDP on RABs / actin filament depolymerization / establishment of protein localization to membrane / intermediate filament / endosomal transport / intercellular bridge / actin filament bundle assembly / antigen processing and presentation / vesicle-mediated transport / cytoskeleton organization / FAD binding / axonogenesis / small monomeric GTPase / G protein activity / secretory granule membrane / negative regulation of protein phosphorylation / monooxygenase activity / protein localization to plasma membrane / Translocation of SLC2A4 (GLUT4) to the plasma membrane / actin filament / adherens junction / trans-Golgi network / cytoplasmic vesicle membrane / cilium / recycling endosome / small GTPase binding / SH3 domain binding / cellular response to insulin stimulus / recycling endosome membrane / phagocytic vesicle membrane / GDP binding / actin filament binding / actin cytoskeleton / actin binding / Factors involved in megakaryocyte development and platelet production / midbody / cytoskeleton / endosome membrane / endosome / Golgi membrane / focal adhesion / Neutrophil degranulation / endoplasmic reticulum membrane / GTP binding / negative regulation of apoptotic process / protein kinase binding / perinuclear region of cytoplasm / Golgi apparatus / signal transduction / extracellular exosome / metal ion binding / plasma membrane / cytosol / cytoplasm

Domain/homology:

DUF3585 / bMERB domain / Bivalent Mical/EHBP Rab binding domain / bMERB domain profile. / LIM zinc-binding domain signature. / LIM domain / Zinc-binding domain present in Lin-11, Isl-1, Mec-3. / Zinc finger, LIM-type / LIM domain profile. / FAD-binding domain / FAD binding domain / ARF-like small GTPases; ARF, ADP-ribosylation factor / small GTPase Rab1 family profile. / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / FAD/NAD(P)-binding domain superfamily / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase

Component:

Ras-related protein Rab-10 / [F-actin]-monooxygenase MICAL1

• Biological species: Homo sapiens (human)
• Citation: Journal: Protein Sci / Year: 2019; Title: Human MICAL1: Activation by the small GTPase Rab8 and small-angle X-ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8.; Authors: Alessandro Esposito / Valeria Ventura / Maxim V Petoukhov / Amrita Rai / Dmitri I Svergun / Maria A Vanoni / ; Abstract: Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD-containing monooxygenase-like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C-terminal helical domain presumably shifting the equilibrium from the free - auto-inhibited - conformation to the active one. We here extend the characterization of the MICAL1-Rab8 interaction and show that indeed Rab8, in the active GTP-bound state, stabilizes the active MICAL1 conformation causing a specific four-fold increase of k of the NADPH oxidase reaction. Kinetic data and small-angle X-ray scattering (SAXS) measurements support the formation of a 1:1 complex between full-length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C-terminal Rab-binding domain may mask one of the Rab-binding sites detected with the isolated C-terminal fragment. SAXS-based modeling allowed us to propose the first model of the free full-length MICAL1, which is consistent with an auto-inhibited conformation in which the C-terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.

Contact author

• Maxim Petoukhov (EMBL-Hamburg, European Molecular Biology Laboratory (EMBL) - Hamburg outstation, Notkestraße 85, Geb. 25A, 22607 Hamburg, Deutschland, Germany)

Models

• Model #2179: ; Type: mix / Radius of dummy atoms: 1.90 A / Chi-square value: 1.28 / P-value: 0.006548; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2180: ; Type: mix / Radius of dummy atoms: 1.90 A / Chi-square value: 1.39 / P-value: 0.000409; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2181: ; Type: mix / Radius of dummy atoms: 1.90 A / Chi-square value: 1.50; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: NADPH oxidase (H2O2 producing and [F-actin] oxidizing) MICAL1 (monomer) in complex with Ras-related protein Rab 8 (MICAL1-Rab8 complex); Specimen concentration: 10 mg/ml / Entity id: 1192 / 1194
• Buffer: Name: 20 mM Hepes/NaOH, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 2 mM DTT; pH: 7.5 / Comment: Mical1:Rab8

Entity #1192

Name: Mical1 / Type: protein / Description: [F-actin]-monooxygenase MICAL1 (monomer) / Formula weight: 117.93 / Num. of mol.: 1 / Source: Homo sapiens / References: UniProt: Q8TDZ2
Sequence:

MASPTSTNPA HAHFESFLQA QLCQDVLSSF QELCGALGLE PGGGLPQYHK IKDQLNYWSA KSLWTKLDKR AGQPVYQQGR ACTSTKCLVV GAGPCGLRVA VELALLGARV VLVEKRTKFS RHNVLHLWPF TIHDLRALGA KKFYGRFCTG TLDHISIRQL QLLLLKVALL LGVEIHWGVT FTGLQPPPRK GSGWRAQLQP NPPAQLANYE FDVLISAAGG KFVPEGFKVR EMRGKLAIGI TANFVNGRTV EETQVPEISG VARIYNQSFF QSLLKATGID LENIVYYKDD THYFVMTAKK QCLLRLGVLR QDWPDTNRLL GSANVVPEAL QRFTRAAADF ATHGKLGKLE FAQDAHGQPD VSAFDFTSMM RAESSARVQE KHGARLLLGL VGDCLVEPFW PLGTGVARGF LAAFDAAWMV KRWAEGAESL EVLAERESLY QLLSQTSPEN MHRNVAQYGL DPATRYPNLN LRAVTPNQVR DLYDVLAKEP VQRNNDKTDT GMPATGSAGT QEELLRWCQE QTAGYPGVHV SDLSSSWADG LALCALVYRL QPGLLEPSEL QGLGALEATA WALKVAENEL GITPVVSAQA VVAGSDPLGL IAYLSHFHSA FKSMAHSPGP VSQASPGTSS AVLFLSKLQR TLQRSRAKEN AEDAGGKKLR LEMEAETPST EVPPDPEPGV PLTPPSQHQE AGAGDLCALC GEHLYVLERL CVNGHFFHRS CFRCHTCEAT LWPGGYEQHP GDGHFYCLQH LPQTDHKKEG SDRGPESPEL PTPSENSMPP GLSTPTASQE GAGPVPDPSQ PTRRQIRLSS PERQRLSSLN LTPDPEMEPP PKPPRSCSAL ARHALESSFV GWGLPVQSPQ ALVAMEKEEK ESPFSSEEEE EDVPLDSDVE QALQTFAKTS GTMNNYPTWR RTLLRRAKEE EMKRFCKAQT IQRRLNEIEA ALRELEAEGV KLELALRRQS SSPEQQKKLW VGQLLQLVDK KNSLVAEEAE LMITVQELNL EEKQWQLDQE LRGYMNREEN LKTAADRQAE DQVLRKLVDL VNQRDALIRF QEERRLSELA LGTGAQG

Entity #1194

Name: Rab8 / Type: protein / Description: Ras-related protein 8 / Formula weight: 20.171 / Num. of mol.: 1 / References: UniProt: P61026
Sequence:

GHMAKKTYDL LFKLLLIGDS GVGKTCVLFR FSDDAFNTTF ISTIGIDFKI KTVELQGKKI KLQIWDTAGQ ERFHTITTSY YRGAMGIMLV YDITNGKSFE NISKWLRNID EHANEDVERM LLGNKCDMDD KRVVPKGKGE QIAREHGIRF FETSAKANIN IEKAFLTLAE DILRKTP

Experimental information

• Beam: Instrument name: PETRA III EMBL P12 / City: Hamburg / 国: Germany / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.124 Å / Dist. spec. to detc.: 3 mm
• Detector: Name: Pilatus 2M

Scan

Title: NADPH oxidase (H2O2 producing and [F-actin] oxidizing) MICAL1 (monomer) in complex with Ras-related protein Rab 8 (MICAL1-Rab8 complex)
Measurement date: Jun 20, 2017 / Storage temperature: 23 °C / Cell temperature: 10 °C / Exposure time: 1 sec. / Number of frames: 3600 / Unit: 1/nm /

	Min	Max
Q	0.1079	5.0354

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 756 /

	Min	Max
Q	0.107882	2.18395
P(R) point	1	756
R	0	11.81

Result

D max: 11.81 / Type of curve: sec
Comments: 1:1 Complex between Mical1 and Rab8. Note: The protein in the SAXS experiments is Rab8, however the protein used for modelling is Rab10.

	Experimental	Porod
MW	142 kDa	142 kDa
Volume	-	234 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	8764	8788	-
Radius of gyration, Rg	3.637 nm	3.7 nm	0.1