Human inositol polyphosphate-5-phosphatase E

Target ID:

INPP5EA

Aliases:

INPP5EMGC117201PPI5PIV

Deposition Date:

Thursday 30th September 2010

Families:

Phosphoinosotide dependent signalling

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2XSW

Authors:

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

2XSW

tabs

Structure Details

Human inositol polyphosphate 5-phosphatases constitute a family of 10 enzymes that hydrolyze the 5-position phosphate from both membrane bound phosphoinositides and soluble inositol phosphates with different specificities, and regulate diverse cellular functions such as vesicle trafficking, Ca2+ signaling, metabolism and cell growth (1). INPP5E dephosphorylates the 5-position of PtdIns(3,4,5)P3 and PtdIns(4,5)P2, and is the sole member of the inositol polyphosphate 5-phosphatase family that in vitro exclusively hydrolyses membrane bound phosphoinositides and is inactive towards soluble inositol phosphates (2). The protein is localized to the cytoplasmic surface of the Golgi and may have a role in phosphoinositide-mediated vesicular trafficking from Golgi (3). INPP5E is implicated in regulation of PI3K-dependent insulin signaling in the hypothalamus. Antisense-mediated reduction of INPP5E in hypothalamus in rats affected metabolic parameters such as insulin and glucose levels and resulted in reduced food intake and weight loss (4). Recent studies indicate that INPP5E also is involved in the primary cilium function. Mice deficient for INPP5E develop a multiorgan disorder associated with structural defects of the primary cilium and in human an INPP5E mutation have been identified that affects cilium stability in a family with MORM syndrome characterized by mental retardation, obesity, congenital retinal dystrophy and micropenis in males (5). In addition, mutations of INPP5E impairing its catalytic activity have been linked to Joubert syndrome, which is another example of a disease for which the underlying cause may be malfunction of primary cilia (6).

We have solved the structure of the catalytic domain of human INPP5E to 1.9 Å and deposited it in the PDB under accession code 2XSW.
The typical fold of the inositol polyphosphate 5-phosphatases appeared to be conserved in this structure with the exception of the C-terminal α-helix which, instead of being rejected far from the protein core, is bridged to a perpendicular α-helix in the close vicinity of the putative substrate-binding site. Additionally, comparison with the structure of INPP5B complexed with a produce (PDB entry 3MTC) revealed important conformational changes in the region supposed to bind the phosphate in position 1 of the substrate. Thus, it is not clear yet if the structure of INPP5E represents an autoinhibited form or if the substrate-binding mode of INPP5E is different from protein of the same family.

Materials and Methods

INPP5E

PDB:2XSW

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC028032
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:INPP5EA-k040
Tag:C-terminal hexahistidine tag
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:MEGSLLASGALLGADELARYFPDRNVALFVATWNMQGQKELPPSLDEFLLPAEADYAQDLYVIGVQEGCSDRREWETRLQETLGPHYVLLSSAAHGVLYMSLFIRRDLIWFCSEVECSTVTTRIVSQIKTKGALGISFTFFGTSFLFITSHFTSGDGKVAERLLDYTRTVQALVLPRNVPDTNPYRSSAADVTTRFDEVFWFGDFNFRLSGGRTVVDALLCQGLVVDVPALLQHDQLIREMRKGSIFKGFQEPDIHFLPSYKFDIGKDTYDSTSKQRTPSYTDRVLYRSRHKGDICPVSYSSCPGIKTSDHRPVYGLFRVKVRPGRDNIPLAAGKFDRELYLLGIKRRISAHHHHHH
Vector:pNIC-CH2

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 60 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 28 °C overnight. The overnight culture (60 ml) was used to inoculate 3 x 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and approximately 200 µl Antifoam 204 (Sigma). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD600 reached ~3. 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 (4,430 x g, 10 min, 4 °C). The resulting cell pellet (115 g wet cell weight) was resuspended in lysis buffer (1.5 ml/g cell pellet), supplemented with 3750 U HS-Nuclease and 3 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 200 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 10.2 mg/ml in a volume of 1.1 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 (10 mg/ml) including 2 mM MgCl2 and 1 mM PtdIns(3,4,5)P3-dioctanoyl was mixed with 0.1 µl of well solution consisting of 0.2 M MgCl2, 0.1 M Tris pH 7.0, 10% PEG 8000. The plate was incubated at 4 ºC and crystals appeared within 5 days. The crystals were quickly transferred to a cryo solution consisting of well solution complemented with 25% glycerol and 1 mM PtdIns(3,4,5)P3-dioctanoyl, and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 1.8 Å resolution was collected at ESRF, beamline ID29.
Data Processing:The structure was solved by molecular replacement using Schizosaccharomyces pombe synaptojanin as a template (PDB entry 1I9Y). The space group was P2₁2₁2₁ with cell dimensions a=50.53 Å, b=103.58 Å and c=137.54 Å. Refinement was performed using iteratice cycles of manual building in Coot and maximum-likelihood refinement in PHENIX. TLS refinement using 3 TLS groups per monomer was employed in the final stages of refinement. At the end of the refinement, R values reached 16.65% and 19.47% for R and R_free, respectively. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 2XSW.

References

Ooms LM, Horan KA, Rahman P, Seaton G, Gurung R, Kethesparan DS, Mitchell CA. (2009) The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease. Biochem J. 419:29-49 PubMed
Kisseleva MV, Wilson MP, Majerus PW. (2000) The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase. J Biol Chem. 275:20110-6 PubMed
Kong AM, Speed CJ, O'Malley CJ, Layton MJ, Meehan T, Loveland KL, Cheema S, Ooms LM, Mitchell CA. (2000) Cloning and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network. J Biol Chem. 275:24052-64 PubMed
Bertelli DF, Araújo EP, Cesquini M, Stoppa GR, Gasparotto-Contessotto M, Toyama MH, Felix JV, Carvalheira JB, Michelini LC, Chiavegatto S, Boschero AC, Saad MJ, Lopes-Cendes I, Velloso LA. (2006) Phosphoinositide-specific inositol polyphosphate 5-phosphatase IV inhibits inositide trisphosphate accumulation in hypothalamus and regulates food intake and body weight. Endocrinology. 147:5385-99 PubMed
Jacoby M, Cox JJ, Gayral S, Hampshire DJ, Ayub M, Blockmans M, Pernot E, Kisseleva MV, Compère P, Schiffmann SN, Gergely F, Riley JH, Pérez-Morga D, Woods CG, Schurmans S. (2009) INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse. Nat Genet. 41:1027-31 PubMed
Bielas SL, Silhavy JL, Brancati F, Kisseleva MV, Al-Gazali L, Sztriha L, Bayoumi RA, Zaki MS, Abdel-Aleem A, Rosti RO, Kayserili H, Swistun D, Scott LC, Bertini E, Boltshauser E, Fazzi E, Travaglini L, Field SJ, Gayral S, Jacoby M, Schurmans S, Dallapiccola B, Majerus PW, Valente EM, Gleeson JG. (2009) Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies. Nat Genet. 41:1032-6 PubMed