Human PIK3R2 (PI3K regulatory subunit beta), RhoGAP domain

Target ID:

PIK3R2A

Aliases:

p85p85-BETAP85BPIK3R2

Deposition Date:

Friday 24th September 2010

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2XS6

Authors:

TrÃ©saugues, L., Arrowsmith, C.H.,Berglund, H.,Bountra, C.,Collins, R.,Edwards, A.M.,Flodin, S.,Flores, A.,Graslund, S.,Hammarstrom, M.,Johansson, I.,Karlberg, T.,Kol, S.,Kotenyova, T.,Kouznetsova, E.,Moche, M.,Nordlund, P.,Nyman, T.,Persson, C.,Schuler, H.,Schutz, P.,Siponen, M.I.,Thorsell, A.G.,Tresaugues, L.,van der Berg, S.,Wahlberg, E.,Welin, M.,Weigelt, J.,Structural Genomics Consortium (SGC)

Site:

Stockholm

2XS6

tabs

Structure Details

Class 1A phosphatidylinositol 3-kinases (PI3Ks) phosphorylate phosphatidylinositol 4,5-bisphosphate and consist of a p110 catalytic subunit and a p85 regulatory subunit (1). In the basal state the regulatory subunit inhibits the kinase activity of the catalytic subunit. In stimulated cells the p85-p110 complex is recruited to phosphorylated tyrosines in activated receptors or adaptor molecules by means of SH2 domains in the p85 subunit. The p85 mediated inhibition of the catalytic activity is thus released and the enzyme is brought in contact with its lipid substrate in the membrane (2). In addition to two SH2 domains the p85α (PIK3R1) and p85β (PIK3R2) isoforms also consist of a SH3 domain and a RhoGAP domain. The function of the RhoGAP domain in the p85α subunit is implicated in the down-regulation of activated receptors through its GAP activity towards several RabGTPases (3). Studies of p85β knockout mice have revealed an important function of this regulatory subunit in B cell development and survival (4).
We have determined the structure of the RhoGAP domain of PIK3R2 to 2.1 Å by molecular replacement using the structure of the corresponding domain of the p85α isoform (PDB entry: 1PBW). The refined model was then deposited in the PDB under accession code 2XS6.
Residues 202, 203 and the region located between residues 256 and 275 were absent of the model due to the lack of electron density in these zones. The latter is probably intrinsically disordered due to its high proline content (eight prolines out of eighteen aminoacids). The overall fold is similar to other RhoGAP domains and could be defined by a bundle of seven α-helices.
Relatively to the previously determined structures of RhoGAP domains, two main conformational changes were observed:

The β-turn located on the C-terminal extremity of the helix E (according to the numbering of Longenecker et al. (5)) is not present. The corresponding region in PIK3R2 RhoGAP domain is rejected up to 7Å towards helix G and, in addition, helix F is one turn longer on its N-terminal side.
The helix A1 present in all RhoGAP domain structures was replaced by a 30 amino acids-long loop which connects directly helix A and helix B. The region which should correspond to the loop connecting helix A to helix A1 is consequently also displaced 7Å closer to the C-terminal side of helix B.

The latter conformational change is of special importance because it rejects the sidechain of Arg147 in a opposite direction relatively to the corresponding residue in other RhoGAP domains. This arginine should be the residue responsible of activating the GTPase partner through transition state stabilization (6). If it still might be possible to attribute such drastic differences to crystal packing, sequence analysis reveals an unusual pattern of prolines in both regions which might constraint PIK3R2 RhoGAP domain in the intrinsically inactive state observed in this structure.

Materials and Methods

PIK3R2

PDB:2XS6

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC070082
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:PIK3R2A-s001
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:mhhhhhhssgvdlgtenlyfq*smGLTLPDLPEQFSPPDVAPPLLVKLVEAIERTGLDSESHYRPELPAPRTDWSLSDVDQWDTAALADGIKSFLLALPAPLVTPEASAEARRALREAAGPVGPALEPPTLPLHRALTLRFLLQHLGRVARRAPALGPAVRALGATFGPLLLRAPPPPSSPPPGGAPDGSEPSPDFPALLVEKLLQEHLEEQEVA
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 (3x15 ml) was used to inoculate 4.5 (3x1.5) 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 (66 g wet cell weight) was resuspended in lysis buffer (1.5 ml/g cell pellet), supplemented with 8000 U Benzonase (Merck) and 4 tablets of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 °C.

Purification

Procedure
Columns
IMAC: Ni-charged 5 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare)

Procedure
IMAC columns were equilibrated with IMAC wash1 buffer, and gel filtration columns were equilibrated with GF buffer. Purification of the protein was performed on an ÄKTAxpress system (GE Healthcare). 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 identified by SDS-PAGE, pooled, and fresh TCEP was added to a final concentration of 2 mM. The protein was concentrated using an Amicon Ultra-15 centrifugal filter device (10,000 NMWL; Millipore) to 13.4 mg/ml in a volume of 0.7 ml. 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 (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 (13.4 mg/ml) was mixed with 0.2 µl of well solution consisting of 0.1 M sodium chloride, 0.1 M sodium cacodylate pH 6.5, 0.85 M tri-sodium citrate dihydrate. The plate was incubated at 20 ºC and crystals appeared within 3 days. The crystals were quickly transferred to a cryo solution consisting of 0.3 M sodium chloride, 0.1 M sodium cacodylate pH 6.5, 0.95 M tri-sodium citrate dihydrate, 25% glycerol and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 2.1 Å resolution was collected at BESSY, beamline BL14-2.
Data Processing:The structure was solved by molecular replacement using the BCR homology domain of the p85 alpha subunit as template (PDB: 1PBW). The space group was C121 with cell dimensions a=94.6 Å b=34.4 Å c=71.4Å, β=130.99. One monomer was present in the asymmetric unit. Refmac5 was used for refinement and Coot for model building. TLS refinement was also used in the last steps using 5 TLS groups per monomer. At the end of the refinement the R values were: R=19.4% and R_free=24.0%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 2XS6.

References

Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B. (2010) The emerging mechanisms of isoform-specific PI3K signalling. Nat. Rev. Mol. Cell Biol. 11:329-41. PubMed
Geering B, Cutillas PR, Vanhaesebroeck B. (2007) Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits? Biochem. Soc. Trans. 35:199-203. PubMed
Chamberlain MD, Berry TR, Pastor MC, Anderson DH. (2004) The p85alpha subunit of phosphatidylinositol 3'-kinase binds to and stimulates the GTPase activity of Rab proteins. J. Biol. Chem. 279:48607-14. PubMed
Oak JS, Chen J, Peralta RQ, Deane JA, Fruman DA. (2009) The p85beta regulatory subunit of phosphoinositide 3-kinase has unique and redundant functions in B cells. Autoimmunity 42:447-58. PubMed
Longenecker KL, Zhang B, Derewenda U, Sheffield PJ, Dauter Z, Parsons JT, Zheng Y, Derewenda ZS. (2000) Structure of the BH domain from graf and its implications for Rho GTPase recognition. J. Biol. Chem. 275:38605-10. PubMed

Rittinger K, Walker PA, Eccleston JF, Smerdon SJ, Gamblin SJ. (1997) Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389:758-62. PubMed