The phox homology domain of human phosphoinositide-3-kinase-C2-gamma

Target ID:

PIK3C2GA

Deposition Date:

Thursday 22nd October 2009

Families:

Phosphoinosotide dependent signalling

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

2WWE

Authors:

Roos, A.K., 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., Kallas, A., Karlberg, T., Kotyenova, T., Kotzch, A., Kraulis, P., Markova, N., Moche, M., Nielsen, T.K., Nyman, T., Persson, C., Schuler, H., Schutz, P., Siponen, M.I., Svensson, L., Thorsell, A.G., van den Berg, S., Wahlberg, E., Weigelt, J., Welin, M., Wisniewska, M., Nordlund, P., Structural Genomics Consortium (SGC)

Site:

Stockholm

2WWE

tabs

Structure Details

PIK3C2G (Phosphoinositide 3-Kinase-C2-gamma) belongs to the class II family of phosphoinositide 3-kinases (PI3Ks) that use preferentially phosphatidylinositol or phosphatidylinositol-4-phosphate as substrates [1,2]. There are three class II PI3K isoforms, and a structural feature that distinguishes this subfamily from other PI3Ks is a C-terminal extension containing a C2 domain and a PX (phox homology) domain. PX domains are found in more than 100 eukaryotic proteins and several act as specific phosphoinositide binding modules with varying lipid-binding specificity [3]. The class II family PI3Ks are often localized to membrane structures and are activated by RTKs, cytokine receptors and integrins [1]. Amplification of the PIK3C2G gene in ovarian cancer cell line indicates that it is a putative oncogene [4]. Moreover SNPs of the PIK3C2G gene has been associated with type II diabetes [5]. However no clear cellular role for this PIK3 family has been defined, partly due to the lack of specific inhibitors.

We have solved the PX domain of PIK3C2G to a resolution of 1.25 Å and deposited the coordinates and structure factors in the protein data bank with the code 2WWE. The structure was solved by molecular replacement using an ensemble of the PX domains 2AR5, 2IWL, 1O7K, 2V6V as the search model. Comparisons to these other PX domains show that the overall fold is very similar despite low sequence homology (24% seq. identity). Main differences are seen in the loop regions connecting the secondary structural elements. Superpositioning the p40phox PX-domain (1H6H.pdb [6]), which has a molecule of phosphatidylinositol 3-phosphate in its ligand binding pocket onto our structure shows that the residues lining the pocket are not conserved. In 1H6H several arginines and a lysine are involved in ligand binding, whereas in the PIK3C2G PX domain the equivalent residues are either negatively charged or hydrophobic, suggesting that this PX domain has a different mode of binding its substrate. Arg 1275 is situated in a favourable position for binding the phosphate moiety of a molecule of phosphatidylinositol-4-phosphate. The membrane interaction loop of PIK3C2G PX is the area of the structure with least ordered density implying higher flexibility in this region. It should also be noted that crystal packing interactions may influence this part of the structure.

Materials and Methods

PIK3C2G

PDB:2WWE

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|194353959, BC130277
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:PIK3C2GA-k023
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Host:E. coli BL21(DE3) R3 pRARE

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMSIERATILGFSKKSSNLYLIQVTHSNNETSLTEKSFEQFSKLHSQLQKQFASLTLPEFPHWWHLPFTNSDHRRFRDLNHYMEQILNVSHEVTNSDCVLSFFLSE
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were used to inoculate 40 mL TB supplemented with 100 µg/mL kanamycin and 34 μg/mL chloramphenicol, and grown at 30 °C overnight. 25 mL of the overnight culture was used to inoculate 1.5 L TB supplemented with 8 g/L glycerol, 50 µg/mL kanamycin and approximately 5-10 drops of Dow Corning Antifoam. The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD600 reached ~2.4. The culture was down-tempered to 18 °C over a period of one 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 (39 g wet cell weight) was resuspended in lysis buffer (1.5 mL/g cell pellet) supplemented with one tablets of Complete EDTA-free protease inhibitor (Roche Applied Science) and 2000 U Benzonase (Merck) per 100 mL lysis buffer, and stored at -80 °C.

Purification

Procedure
Columns
IMAC: Ni-charged 2 x 1 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare)

Procedure
Purification of the native 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 the Ni-charged HiTrap Chelating column 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 subsequently concentrated using an Amicon Ultra-15 centrifugal filter device, 5 000 NMWL (Millipore). The concentration was measured to 26.4 mg/mL in a volume of 1.2 ml and the samples were stored at -80 ºC. The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water. Cells were disrupted by sonication (VibraCell, Sonics) at 80% amplitude for 3 min effective time (puls 4s on, 4s off) and cell debris was removed by centrifugation (49,000 x g, 30 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
MassSpec:
Crystallization:Native crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.2 µl of the protein solution (26.4 mg/ml) was mixed with 0.1 µl of well solution consisting of 0.1M Tri-sodium citrate dihydrate pH 5.5, 20% PEG3000. The plate was incubated at 4 ºC. Crystals of dimensions 75x75 µm had appeared after 5 days and grew to a final size of 125x125 µm within two weeks. The crystals were transferred to cryo solution containing 0.1M Tri-sodium citrate dihydrate pH 6.5, 22% PEG3000, 20% Glycerol, 0.3M NaCl, and flash cooled in liquid nitrogen prior to data collection.
NMR Spectroscopy:
Data Collection:Native data was collected to 1.25 Å at DIAMOND (I04). The crystal belonged to space group P 32 2 1 with cell parameters of a=53.42 Å b=53.42 Å and c=75.13 Å.
Data Processing:Data were integrated in iMosflm and scaled with Scala. The structure was solved by molecular replacement in Phaser using a search model consisting of an ensemble of the superpositioned coordinates from Phox domains 2AR5, 2IWL, 1O7K, 2V6V. ArpWarp could be used for automatic model building of the one molecule present in the asymmetric unit. Manual rebuilding was performed in Coot and cycled with restrained refinement in REFMAC5, which resulted in the final model where R= 16.5% and Rfree=18.4%. The coordinates and structure factors were deposited in the PDB with accession code 2WWE.

References

Falasca M, Maffucci T. (2007). Role of class II phosphoinositide 3-kinase in cell signalling. Biochem Soc Trans. 35(Pt 2):211-4.
PubMed 17371240
Rozycka M, Lu YJ, Brown RA, Lau MR, Shipley JM, Fry MJ. (1998). cDNA cloning of a third human C2-domain-containing class II phosphoinositide 3-kinase, PI3K-C2gamma, and chromosomal assignment of this gene (PIK3C2G) to 12p12. Genomics. 54(3):569-74.
PubMed 9878262
Ellson CD, Andrews S, Stephens LR, Hawkins PT. (2002). The PX domain: a new phosphoinositide-binding module. J Cell Sci. 115(Pt 6):1099-105.
PubMed 11884510
Lambros MB, Fiegler H, Jones A, Gorman P, Roylance RR, Carter NP, Tomlinson IP. (2005). Analysis of ovarian cancer cell lines using array-based comparative genomic hybridization. J Pathol. 205(1):29-40.
PubMed 15586366
Daimon M, Sato H, Oizumi T, Toriyama S, Saito T, Karasawa S, Jimbu Y, Wada K, Kameda W, Susa S, Yamaguchi H, Emi M, Muramatsu M, Kubota I, Kawata S, Kato T. (2008). Association of the PIK3C2G gene polymorphisms with type 2 DM in a Japanese population. Biochem Biophys Res Commun. 365(3):466-71.
PubMed 17991425
Bravo J, Karathanassis D, Pacold CM, Pacold ME, Ellson CD, Anderson KE, Butler PJ, Lavenir I, Perisic O, Hawkins PT, Stephens L, Williams RL. (2001). The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate. Mol. Cell 8(4):829-39.
PubMed 11684018