Human SEC14L3 SEC14-like 3

Target ID:

SEC14L3C

Deposition Date:

Tuesday 2nd September 2014

Families:

Miscellaneous

Related Diseases:

Metabolism

Structure type:

protein-ligand

Download Coordinates:

4UYB

Authors:

J.Kopec,S.Goubin,T.Krojer,N.Burgess-Brown,F. von Delft,C.Arrowsmith,A.Edwards,C.Bountra,W.W.Yue

Site:

Oxford

4UYB

tabs

Structure Details

Protein-lipid interactions are very important for cell membrane maintenance, lipid transport and metabolism. SEC14 domains have been shown to bind lipids and transport them to the target membranes [1]. A subset of SEC14 domain containing proteins, termed SEC14L1-5, contain a GOLD (Golgi dynamics) domain in addition to the SEC14 domain [2]. GOLD domains are thought to act as protein-protein interaction modules involved in Golgi function and trafficking [2]. SEC14L3C has been shown to be involved in airway inflammation, as there is an inverse relationship between SEC14L3C mRNA/protein expression and allergic airway inflammation [3].
Binding of lipids to SEC14L2 and SEC14L3 is much better characterized than to other SEC14L proteins [4]. SEC14L2 binds α-, β-,γ- and δ-tocopherols, α-tocopherol quinone, tocopherol succinate, squalene, PtdCHo and PtdIns in vitro, but it is unclear which of these are physiological ligands. SEC14L2 takes part in vitamin E transport, assistance in squalene epixidation to lanosterol for cholesterol biosynthesis and stimulation ofHMG-CoA reductase. SEC14L3 seems to have different lipid ligands and physiological function than SEC14L2. It binds PtdIns(3,4,5)P3 and co-localizes with this lipid in cells. It accumulates in secretory vesicles and in the extracellular matrix, which means that SEC14L3 exports lipids out of the cells. SEC14L4 can bind in vitro the same lipids as SEC14L2 and SEC14L3[5].
SEC14L2-4 have been shown to hydrolyze GTP, which might modulate the ligand binding, localization within the cell or the interaction with other proteins [6]. GTP and GDP binding might also cause structural changes in SEC14L proteins leading to active/inactive state. Here, we have determined the structure of the full-length SEC14L3C protein to 1.50 Å resolution.

Materials and Methods

SEC14L3

PDB:4UYB

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC101004 (IMAGE:40006377)
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal, TEV protease cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMSGRVGDLSPKQAETLAKFRENVQDVLPALPNPDDYFLLRWLRARNFDLQKSEALLRKYMEFRKTMDIDHILDWQPPEVIQKYMPGGLCGYDRDGCPVWYDIIGPLDPKGLLFSVTKQDLLKTKMRDCERILHECDLQTERLGKKIETIVMIFDCEGLGLKHFWKPLVEVYQEFFGLLEENYPETLKFMLIVKATKLFPVGYNLMKPFLSEDTRRKIIVLGNNWKEGLLKLISPEELPAQFGGTLTDPDGNPKCLTKINYGGEIPKSMYVRDQVKTQYEHSVQINRGSSHQVEYEILFPGCVLRWQFSSDGADIGFGVFLKTKMGERQRAGEMTEVLPSQRYNAHMVPEDGNLTCSEAGVYVLRFDNTYSFVHAKKVSFTVEVLLPDEGMQKYDKELTPV

MHHHHHHSSGVDLGTENLYFQ*SM is the purification tag plus TEV protease recognition site *.
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure
Buffers Used:
Binding/Lysis Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP
Wash Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.5mM TCEP
Elution Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.5mM TCEP
Gel Filtration Buffer: 10 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 0.5mM TCEP
Cell Lysis
Cell pellets were resuspended in 4 mL lysis buffer per gram of cell pellet and lysed by passing through the constant cell disruptor at a constant pressure of 15KPa. The cell debris were pelleted at 35 000 g and the supernatant used for further purification.

Column 1
Ni-NTA (2 mL volume in a gravity-flow column).
The clarified cell extract was incubated with 4 mL pre-equilibriated 50% Ni_NTA bead suspension for 1 hour at 4°C with rotation. The suspension was centrifuged at 900g for 10 min. The supernatant was poured away and the beads were resuspended in lysis buffer and transferred onto a gravity column. The column was then washed with 20ml Binding Buffer (2 x 10ml) and 20 ml Wash Buffer (2 x10 ml). The protein was eluted with 10 ml of elution Buffer in 5 x 2 ml fractions.

Column 2
Superdex s200 16/60 Gel Filtration.
Elution and wash fractions containing the target protein were concentrated to 5 mL and applied the size exclusion chromatography column, s200 (pre-equilibriated in GF buffer) at 1.0 ml/min. 1.0 ml fractions were collected.

Enzymatic treatment and purification
The N-terminal His6- tag was cleaved by incubating overnight with TEV at 4°C. Cleaved protein was purified by batch binding on 1ml pre-equilibriated 50% Ni-NTA bead solution. The column was then washed with 2x1ml Gel Filtration buffer,2x1ml Binding buffer, 2x1ml Wash buffer, and finally 2x1ml of Elution buffer.

Column 3
The protein was diluted 10 fold with buffer A (25mM Tris pH 8.5, 5% glycerol, 0.5mM TCEP) and loaded onto 1 mL HiTrapQ column. The protein was eluted in gradient to 1M NaCl.

Extraction

Buffers
Procedure
Expression strain
BL21(DE3)-R3-pRARE2

A glycerol stock was used to inoculate 60 ml of LB media containing 50ug/ml kanamycin and 50 ug/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to inoculate 6L of TB media (7 ml starter culture used per 1L) containing 50 ug/ml kanamycin. When the OD600 reached approximately 0.6 the temperature was reduced to 18°C and after a further 30 minutes the cells were induced by the addition of 0.1 mM IPTG.
Expression was continued overnight.

Cell harvest
Cells were harvested by centrifugation at 6000g after which the media were poured out and the cell pellet either placed in a -80°C freezer or used directly for purification.
Concentration:To set up plates the sample was concentrated to 11.5 mg/ml using a 10 kDa mwco concentrator.
Ligand
MassSpec:Expected mass: 46149.4 Da
Measured mass: 46151.1 Da
Crystallization:Crystals were grown by vapour diffusion in sitting drop at 20°C. A sitting drop consisting of 100 nl protein and 50nl well solution was equilibrated against well solution containing 20% PEG6000, 0.8M lithium chloride and 0.1M citrate pH 4.2
NMR Spectroscopy:
Data Collection:Resolution: 1.45 Å
X-ray source: Diamond Light Source beamline IO3
Data Processing:

References

1. Saito K, Tautz L, Mustelin T. The lipid-binding SEC14 domain. Biochim Biophys Acta. 2007 Jun;1771(6):719-26.

2. Anantharaman V, Aravind L. The GOLD domain, a novel protein module involved in Golgi function and secretion. Genome Biol. 2002;3(5):research0023. Epub 2002 Apr 24. PubMed PMID: 12049664; PubMed Central PMCID: PMC115225.

3. Shan L, Kawakami T, Asano S, Noritake S, Yoshimoto D, Yamashita K, Kikkawa H, Kinoshita M, Matsubara S. Inverse relationship between Sec14l3 mRNA/protein expression and allergic airway inflammation. Eur J Pharmacol. 2009 Aug 15;616(1-3):293-300.

4. C. Panagabko, S. Morley, M. Hernandez, P. Cassolato, H. Gordon, R.Parsons, D. Manor, J. Atkinson, Ligand specificity in the CRAL-TRIO protein family, Biochemistry 42 (2003) 6467-6474

5. P. Kempna, J.M. Zingg, R. Ricciarelli, M. Hierl, S. Saxena, A. Azzi, Cloning of novel human SEC14p-like proteins: ligand binding and functional properties, Free Radic. Biol. Med. 34 (2003) 1458-1472.

6. Habermehl, P. Kempna, A. Azzi, J.M. Zingg, Recombinant SEC14-like proteins (TAP) possess GTPase activity, Biochem. Biophys. Res. Commun. 326 (2005) 254-59