Phosphoinositide-3-kinase, class 3

Target ID:

PIK3C3A

Aliases:

MGC61518PIK3C3Vps34

Deposition Date:

Friday 31st July 2009

Families:

Non-protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

3IHY

Authors:

SIPONEN, M.I., TRESAUGUES, L., ARROWSMITH, C.H., BERGLUND, H., BOUNTRA, C., COLLINS, R., EDWARDS, A.M., FLODIN, S., FLORES, A., GRASLUND, S., HAMMARSTROM, M., JOHANSSON, A., JOHANSSON,I., KARLBERG,T., KOTENYOVA, T., KOTZSCH, A., KRAGH NIELSEN, T., MARKOVA, N., MOCHE,M., NYMAN, T., PERSSON, C., ROOS, A., SAGEMARK, J., SCHUTZ P., SCHUELER, H., THORSELL, A.G., VAN DEN BERG, S., WAHLBERG, E., WEIGELT, J., WELIN, M., WISNIEWSKA, M., NORDLUND, P.

Site:

Stockholm

3IHY

tabs

Structure Details

Phosphatidylinositol (PtdIns) 3-kinases (PI3K) are enzymes that catalyse phosphorylation of the 3′ hydroxyl position of myo-inositol lipids. The enzymes can be divided into three major classes depending on their substrate specificity and subunit organisation [1]. PIK3C3 also named Vps34 (vacuolar protein sorting 34) is the sole member of the class III PI3K and consists of an N-terminal C2 domain, an accessory helical domain and a catalytic domain in the C-terminus. The substrate specificity of PIK3C3 is restricted to phosphatidylinositol and is thus distinguished from the other family members which also use phosphorylated derivatives of this phospholipid as substrate [2]. PIK3C3 is involved in regulation of vesicular trafficking in the endosomal system where the phosphatidylinositol triphosphate (PtdIns3P) produced acts to recruiting proteins that contains PtdIns3P binding domain [3]. PIK3C3 also regulates autophagy [4] and is implicated in neurodegenerative disease and in tumor suppression by having a role in the clearance of pathological protein aggregations. Furthermore PIK3C3 is implicated to have a role in regulating cellular response to nutrient deprivation [5].

Although only weak homology exists and the substrate binding region is unique for PIK3C3, the overall structure bears striking similarities to other PI3 kinases. The accessory domain folds as an α-helical structure. The catalytic domain consists of an N-terminal lobe and a C-terminal lobe separated by a cleft that forms the catalytic site of the enzyme, representing the typical architecture of protein and lipid kinases.

Materials and Methods

PIK3KC3

PDB:3IHY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|31657191, BC033004
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:PIK3C3A-k026
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Host:E.coli BL21(DE3) R3 pRARE, where R3 denotes a derivative of BL21(DE3) resistant to a strain of T1 bacteriophage (SGC Oxford) and the pRARE plasmid originating from the Rosetta strain (Novagen) supplies tRNAs for rare codons.

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMSDHDLKPNAATRDQLNIIVSYPPTKQLTYEEQDLVWKFRYYLTNQEKALTKFLKCVNWDLPQEAKQALELLGKWKPMDVEDSLELLSSHYTNPTVRRYAVARLRQADDEDLLMYLLQLVQALKYENFDDIKNGLEPTKKDSQSSVSENVSNSGINSAEIDSSQIITSPLPSVSSPPPASKTKEVPDGENLEQDLCTFLISRACKNSTLANYLYWYVIVECEDQDTQQRDPKTHEMYLNVMRRFSQALLKGDKSVRVMRSLLAAQQTFVDRLVHLMKAVQRESGNRKKKNERLQALLGDNEKMNLSDVELIPLPLEPQVKIRGIIPETATLFKSALMPAQLFFKTEDGGKYPVIFKHGDDLRQDQLILQIISLMDKLLRKENLDLKLTPYKVLATSTKHGFMQFIQSVPVAEVLDTEGSIQNFFRKYAPSENGPNGISAEVMDTYVKSCAGYCVITYILGVGDRHLDNLLLTKTGKLFHIDFGYILGRDPKPLPPPMKLNKEMVEGMGGTQSEQYQEFRKQCYTAFLHLRRYSNLILNLFSLMVDANIPDIALEPDKTVKKVQDKFRLDLSDEEAVHYMQSLIDESVHALFAAVVEQIH
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 15 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 30 °C overnight. The overnight culture (15 ml) was used to inoculate 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and approximately 200 µl 204 Antifoam A6426 (Sigma). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD600 reached ~2. The culture was down-tempered to 18 °C over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,400 x g, 10 min, 4 °C). The resulting cell pellet (42 g wet cell weight) was resuspended in lysis buffer (1.5 ml/g cell pellet), supplemented with 2000 U Benzonase (Merck) and one tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 °C.

Purification

Procedure
Columns
IMAC: Ni-charged 1 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 200 Prep Grade (GE Healthcare)

Procedure
Purification of the protein was performed as a two step process on an ÄKTAxpress system (GE Healthcare). Prior to purification, columns were equilibrated with IMAC wash1 buffer and gel filtration buffer, respectively. The filtered lysate was loaded onto two Ni-charged HiTrap Chelating columns connected in series and washed with IMAC wash1 buffer followed by IMAC wash2 buffer. Bound protein was eluted from the IMAC column with IMAC elution buffer and automatically loaded onto the gel filtration column. Fractions containing the target protein were pooled and fresh TCEP was added to a final concentration of 2 mM. The total volume of the pooled fractions was 10 ml and the concentration of the protein 0.90 mg/ml. The identity of the protein was confirmed by mass spectrometry.

Tag removal
The N-terminal histidine tag was proteolytically removed by incubating the target protein with His-tagged TEV protease in a molar ratio of 50:1 at 8 ºC overnight. The proteolytic reaction went to completion, as judged by SDS-PAGE. Target protein was purified from tag and protease by passing the reaction mixture over a Ni-charged 1 ml HisTrap HP column (GE Healthcare) pre-equilibrated with IMAC wash1 buffer. The protein was concentrated and the buffer was changed to GF buffer with 2 mM TCEP using a Vivaspin 20 centrifugal filter device with 30,000 MWCO (Sartorius). The final protein concentration was determined to 18.6 mg/ml in a volume of 0.3 ml The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water bath. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,000 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.1 µl protein solution (18.6 mg/ml) supplemented with AMP-PNP (2.5 mM final solution) was mixed with 0.1 µl of well solution consisting of 0.2 M ammonium acetate, 0.1 M tris pH 8.5 and 25% PEG 3350. The plate was incubated at 20 ºC and crystals appeared in less than 14 days. The crystals were quickly transferred to a cryo solution consisting of well solution with PEG 3350 concentration increased to 26% and complemented with 20% glycerol and 0.3 M NaCl, and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 2.8 Å resolution was collected at ESRF beamline ID 14-2.
Data Processing:Data was integrated using XDS and scaled using SCALA. The structure was solved by molecular replacement using PHASER with the phosphatidylinositol 3-kinase catalytic subunit as template (PDB: 1E7U). The space group was P1 with cell dimensions a= 62.30 Å b=96.49 Å c=150.68 Å, α=107.77° β=91.75° γ=92.41°. Five monomers were located in the asymmetric unit. The initial model was improved using PHENIX.AUTOBUILD. The first cycles of refinement were performed with PHENIX.REFINE, while REFMAC5 was used for final refinement and Coot for manual model building. Data in the interval 19.55-2.80 Å resolution was used and at the end of the refinement the R values were: R=18.5% and Rfree=25.2%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 3IHY.

References

Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT, Heintz N, Yue Z. (2009). Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat Cell Biol. 4:468-76.
Byfield MP, Murray JT, Backer JM, (2005). hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase. J Biol Chem 38:33076-82.
Backer JM (2008). The regulation and function of Class III PI3Ks: novel roles for Vps34. Biochem J. 1:1-17.
Volinia S, Dhand R, Vanhaesebroeck B, MacDougall LK, Stein R, Zvelebil MJ, Domin J, Panaretou C, Waterfield MD. (1995). A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system. EMBO J 14:3339-48.
Djordjevic S, Driscoll PC (2002). Structural insight into substrate specificity and regulatory mechanisms of phosphoinositide 3-kinases. Trends Biochem Sci. 8:426-32.