Crystal structure of human diphosphoinositol polyphosphate phosphohydrolase 3-alpha

Target ID:

NUDT10A

Aliases:

APS2DIPP3ahDIPP3alphaNUDT10

Deposition Date:

Monday 29th March 2010

Families:

Nucleotide metabolismNUDIX

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

3MCF

Authors:

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

Site:

Stockholm

3MCF

tabs

Structure Details

NUDIX hydrolases are enzymes which catalyse the cleavage of phosphodiester bonds [1]. Founding member of this family, bacterial MutT, hyrdolyzes 8-oxo-dGTP into 8-oxo-dGMP and pyrophosphate which i) has provided the name of the family (Nucleoside diphosphate linked to an other moiety X hydrolase) and ii) has conferred to the NUDIX protein a role of "house-cleaning enzymes" expected to remove potentially deleterious metabolites [2]. In addition to the canonical activity towards nucleoside diphosphate, some NUDIX can also be active in hydrolyzing diphosphoinositol polyphosphate [3]. Human NUDT10 (hDIPP3α, hAps2) and NUDT11 (hDIPP3β, hAps1) belongs to this latter subfamily [4].

They are encoded by two consecutive genes located on chromosome X and are principally expressed in testis. The two isoforms differ only by the residue in position 89 (Pro and Arg for respectively NUDT10 and NUDT11). They are able to catalyze the hydrolysis of the β-phosphate from diphosphoinositol pentakisphosphate (PP-InsP5) to produce inositol hexakisphosphate (InsP6). NUDT11 appeared to be 2.3-fold more active than NUDT10 with kcat/Km towards PP-InsP5 of 18x105M-1s-1 and 42x105M-1s-1 for respectively NUDT10 and NUDT11. Ability to hydrolyze both diadenosine 5',5'''-pentaphosphate (Ap5A) and diadenosine 5',5'''-hexaphosphate (Ap6A) has been shown for both enzymes and required divalent cations, especially Mn2+ [5]. No significant difference between the two isoforms was noticed when either Ap5A or Ap6A were used as substrates.

Here we present the crystal structure refined to 2.0Å resolution of a construct of human NUDT10 encompassing residues 17 to 144 (PDB entry: ). The structure was solved by molecular replacement using a model base upon the mouse orthologous MS0616 (PDB entry: 2DUK). Two monomers were present in the asymmetric unit and the monomeric state of NUDT10 was also confirmed by gel-filtration. NUDT10 adopts the NUDIX α/β fold which is constituted by a four-stranded mixed β-sheet sandwiched between the α-helix which carries the NUDIX signature (GX5EX7REUXEEXGU where U is a bulky hydrophobic aminoacid and X could be any aminoacid) one one side, and two parallel α-helices on the other side. An additional two stranded antiparallel β-sheet is inserted between the structural secondary elements of the core.

With the exception of residues 85 to 90, all aminoacids were positioned in the electronic density maps. That even includes the C-terminal expression tag which add one more turn to the C-terminus α-helix of chain A. One citrate ion present in the crystallization conditions could also be unambiguously located in each monomer. Its position mimics the one of InsP6 found in the structure of NUDT3 (DIPP-1) with the γ-carboxylate of the citrate being involved in a salt-bridge interaction with Arg115 [6]. In the structure of the complex between NUDT3, InsP6 and Mg-F (PDB entry: 2Q9P), the corresponding position is occupied by a phosphate of InsP6. In addition, the active sites of both NUDT10 and NUDT3 are perfectly conserved including conservation of structural water molecules. The major difference is a changing of main-chain orientation of the N-terminus of NUDT10 which bring away Lys17 from the InsP6 binding-site. In the structure of NUDT3 in complex with the product of the reaction, this residue is involved in capping the negative charge of a phosphate moiety.

The residue which differs between NUDT10 and NUDT11 is located in the disordered region. It corresponds to Arg89 in the structure of NUDT3 and is involved in charge neutralization of InsP6. Thus it is very likely that this region becomes ordered upon ligand binding and that the lack of a positive charge in this region makes binding of PP-InsP5 less favorable in the case of NUDT10 compared to NUDT11.

Materials and Methods

NUDT10

PDB:3MCF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC050700
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:NUDT10A-s001
Tag:C-terminal hexahistidine tag starting with an additional alanine
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:MKKRAACLCFRSEREDEVLLVSSSRYPDRWIVPGGGMEPEEEPGGAAVREVYEEAGVKGKLGRLLGVFEQNQDPEHRTYVYVLTVTELLEDWEDSVSIGRKREWFKVEDAIKVLQCHKPVHAEYLEKLKahhhhhh
Vector:pNIC-CH2

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 70 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 30 °C overnight. The overnight culture (70 ml) was used to inoculate 3 bottles of 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and approximately 200 µl 204 Antifoam A6426 (Sigma). Cultures were grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD₆₀₀ reached ~2. Cultures were 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 (117.9 g wet cell weight) was resuspended in lysis buffer (1.5 ml/g cell pellet), supplemented with 6000 U Benzonase (Merck) and three 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 (5,000 NMWL; Millipore) to 13.2 mg/ml in a volume of 0.2 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. 0.2 µl protein solution (13.2 mg/ml) was mixed with 0.1 µl of well solution consisting of 0.01 M Na acetate trihydrate pH 4.6, 0.1 M tri-Na citrate dihydrate pH 5.5, 0.12 M tri-Na citrate dihydrate unbuffered and 19.8% w/v PEG3000 . The plate was incubated at 4 ºC and crystals appeared within 5 days. The crystals were quickly transferred to a cryo solution consisting of 21% w/v PEG3000, 0.22 M tri-Na citrate dihydrate pH 5.5, 0.01 M Na acetate trihydrate pH 4.6, 25% glycerol, and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 2.0 Å resolution was collected at BESSY, beamline BL14.2.
Data Processing:The structure was solved by molecular replacement using as template a model produced by Swiss-Model based upon MS0616 (PDB: 2DUK). The space group was P212121 with cell dimensions a=56.39 Å b=79.55 Å c=88.73 Å. After automatic model building carried on in ArpWarp, two monomers were located in the asymmetric unit. Iterative cycles of manual model building in Coot and refinement in PHENIX led to the final model. Data in the interval 32.5-2.0 Å resolution was used and at the end of the refinement the R values were: R=17.5% and Rfree=21.7%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 3MCF.

References

McLennan A. The Nudix hydrolase superfamily. Cell Mol Life Sci, 2006, 63(2): p. 123-143. PubMed
Bessman MJ, Frick DN, O'Handley SF. The MutT Proteins or "Nudix" Hydrolases, a Family of Versatile, Widely Distributed, "Housecleaning" Enzymes. J Biol Chem, 1996, 271(41): p. 25059-25062. PubMed
Safrany ST, Caffrey JJ, Yang X, Bembenek ME, Moyer MB, et al. A novel context for the 'MutT' module, a guardian of cell integrity, in a diphosphoinositol polyphosphate phosphohydrolase. EMBO J, 1998, 17(22): p. 6599-6607. PubMed
Hidaka K, Caffrey JJ, Hua L, Zhang T, Falck JR, et al. An Adjacent Pair of Human NUDT Genes on Chromosome X Are Preferentially Expressed in Testis and Encode Two New Isoforms of Diphosphoinositol Polyphosphate Phosphohydrolase. J Biol Chem, 2002, 277(36): p. 32730-32738. PubMed
Leslie N, McLennan A, Safrany S. Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases. BMC Biochem, 2002, 3(1) : p. 20. PubMed
Thorsell A, Persson C, Gräslund S, Hammarström M, Busam RD, et al. Crystal structure of human diphosphoinositol phosphatase 1. Proteins, 2009, 77(1): p. 242-246. PubMed