Human isoprenoid synthase domain containing

Target ID:

ISPDA

Deposition Date:

Thursday 27th March 2014

Families:

Miscellaneous

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

4CVH

Authors:

J.Kopec,D.S.Froese,T.Krojer,J.Newman,W.Kiyani,S.Goubin,C.Strain-Damerell,M.Vollmar,F.von Delft,N.Burgess-Brown,C.Arrowsmith,A.Edwards,C.Bountra,D.J.Lefeber,W.W.Yue

Site:

Oxford

4CVH

tabs

Structure Details

Protein glycosylation is an essential and well-studied post-translational modification that requires the sequential addition of single monosaccharides to form a glycan structure, in which nucleotide- and dolichol-linked sugars serve as donors. Most nucleotide sugars are produced in the cytoplasm from phosphorylated monosaccharides, involving the family of cytidyltransferase enzymes which catalyse the addition of nucleotide to a sugar phosphate group [1].
α-Dystroglycan (α-DG, encoded by DAG1) is a glycoprotein with a unique and not yet fully elucidated O-mannose linked glycan that is crucial for binding extracellular ligands. Defective α-DG O-mannosylation leads to a group of human disorders, known as dystroglycanopathies, which present with muscular dystrophy, severe brain abnormalities and in some cases eye abnormalities [2]. These disorders can be caused by defects in the DAG1 gene itself or, in the majority of cases, its glycosylation enzymes. Recently, mutations in the isoprenoid synthase domain-containing (ISPD) gene were identified in patients with deficient α-DG O-mannosylation [3]. ISPD encodes a protein with sequence conservation to cytidyltransferases. While ISPD homologs in plants, protozoa and some bacteria function to synthesize 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP as part of the non-mevalonate or MEP pathway of isoprenoid synthesis, this pathway is not present in human. As such, the functional role of human ISPD (hISPD) remains unknown, although its homology to cytidyltransferases suggests a possible role in novel nucleotide-sugar synthesis.
To explore the function of human ISPD, we determined its crystal structure in the apo form to 2.7 Å resolution, revealing a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain absent in cytidyltransferase homologues.

Materials and Methods

ISPD

PDB:4CVH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_001101426.1
Entry Clone Source:Collaborator
SGC Clone Accession:
Tag:N-terminal, TEV protease cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:MGHHHHHHSSGVDLGTENLYFQSMHPQAVAAVLPAGGCGERMGVPTPKQFCPILERPLISYTLQALERVCWIKDIVVAVTGENMEVMKSIIQKYQHKRISLVEAGVTRHRSIFNGLKALAEDQINSKLSKPEVVIIHDAVRPFVEEGVLLKVVTAAKEHGAAGAIRPLVSTVVSPSADGCLDYSLERARHRASEMPQAFLFDVIYEAYQQCSDYDLEFGTECLQLALKYCCTKAKLVEGSPDLWKVTYKRDLYAAESIIKERISQEICVVMDTEEDNKHVGHLLEEVLKSELNHVKVTSEALGHAGRHLQQIILDQCYNFVCVNVTTSDFQETQKLLSMLEESSLCILYPVVVVSVHFLDFKLVPPSQKMENLMQIREFAKEVKERNILLYGLLISYPQDDQKLQESLRQGAIIIASLIKERNSGLIGQLLIA

MHHHHHHSSGVDLGTENLYFQ*SM is the purification tag plus TEV protease recognition site *.
Vector:pFB-LIC-Bse Baculovirus transfer vector (Bac-to-bac)

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Binding/Lysis Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP Wash Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.5mM TCEP Elution Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.5mM TCEPGel Filtration Buffer: 10 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 0.5mM TCEP
Procedure
Cell Lysis
Cell pellets were resuspended in 50 mL lysis buffer. The cells were lysed by sonication for 7.5 min 5sec on/ 5 sec off. The cell debris were pelleted at 35 000 g and the supernatant used for further purification.

Column 1
Ni-NTA (1.25 ml volume in a gravity-flow column).
The clarified cell extract was incubated with 5.0 ml pre-equilibriated 50%. Ni_NTA bead suspension for 1 hour at 4°C with rotation. The suspension was centrifuged at 900g for 5 min. The supernatant was poured away and the beads were resuspended in lysis buffer and transferred onto a gravity column. The column was then washed with 30ml Binding Buffer (2 x 15ml) and 50 ml Wash Buffer (2 x15 ml). The protein was eluted with 25 ml of elution Buffer in 5 x 2.5 ml fractions.

Column 2
Superdex s200 16/60 Gel Filtration.
Elution fraction 1&2 were concentrated to 5 mL and applied directly to the size exclusion chromatography column, s200 (pre-equilibriated in GF buffer).

Enzymatic treatment and purification
The N-terminal His6- tag was cleaved by incubating overnight with TEV (20°C). Cleaved protein was purified by batch binding on 125uL pre-equilibriated 50% Ni-NTA bead solution. The column was then washed with 2x500uL Gel Filtration buffer,2x500uL Binding buffer, 2x500uLWash buffer, and finally 2x500uL of Elution buffer.

Extraction

Buffers
Procedure
Expression strain

Expression system: BEVS
Host cell line: Sf9
Expression medium: Insect-Xpress
Cell density at the time of infection: 2 x 10E6 cells/ml
Expression temperature: 27°C
Virus: used 3 ml of P2/L (unless stated otherwise)
Expression time point: ~ 70-72 hrs
Culture vessel: 3L glass flasks without baffles
Culture volume: Each 3 L flask contained 1L
Total volume 6L

Cell harvest
Cells were harvested by centrifugation at 900g after which the media were poured out and the cell pellet placed in a -80°C freezer.
Concentration:To set up plates the sample was concentrated to 14.76 mg/ml using a 10 kDa mwco concentrator.
Ligand
MassSpec:Expected mass: 46049.6 Da
Measured mass: 46048.96 Da
Crystallization:Crystals were grown by vapour diffusion in sitting drop at 20°C. A sitting drop consisting of 50 nl protein and 100nl well solution was equilibrated against well solution containing 0.1M bis-tris pH 6.5, 25%(w/v) PEG 3350.
NMR Spectroscopy:
Data Collection:Resolution: 2.4 Å
X-ray source: Diamond Light Source beamline I03
Data Processing:

References

[1] Bulter T, Elling L (1999) Enzymatic synthesis of nucleotide sugars. Glycoconj J 16: 147-159

[2] Martin PT (2005) The dystroglycanopathies: the new disorders of O-linked glycosylation. Seminars in pediatric neurology 12: 152-158

[3] Roscioli T, Kamsteeg EJ, Buysse K, Maystadt I, van Reeuwijk J, van den Elzen C, van Beusekom E, Riemersma M, Pfundt R, Vissers LE, Schraders M, Altunoglu U, Buckley MF, Brunner HG, Grisart B, Zhou H, Veltman JA, Gilissen C, Mancini GM, Delree P, Willemsen MA, Ramadza DP, Chitayat D, Bennett C, Sheridan E, Peeters EA, Tan-Sindhunata GM, de Die-Smulders CE, Devriendt K, Kayserili H, El-Hashash OA, Stemple DL, Lefeber DJ, Lin YY, van Bokhoven H (2012) Mutations in ISPD cause Walker-Warburg syndrome and defective glycosylation of alpha-dystroglycan. Nat Genet 44: 581-585