Human AD-003 protein

Target ID:

AD-003

Aliases:

AD-003

Deposition Date:

Monday 7th November 2005

Families:

Transferases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2EX4

Authors:

J.R.MIN,H.WU,H.ZENG,P.LOPPNAU,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2EX4

tabs

Structure Details

The methylation of a variety of biologically active molecules is a common theme in biology.
The functional roles of methylation are wide ranging and include biosynthesis, metabolism, detoxification, signal transduction, protein sorting and repair, nucleic acid processing, gene silencing and imprinting.
The majority of methylation reactions are carried out by the S-adenosyl- L-methionine (SAM)-dependent methyltransferases.

We have solved human putative methyltransferase AD-003 crystal structure in complex with S-adenosyl-L-homocysteine at 1.75 Å resolution. AD-003 gene is located on human chromosome 9q34.11.
AD-003 protein belongs to a family of protein with unknown function (DUF858) that are SAM-dependent methyltransferase-like. It is widely expressed in human tissues including brain, heart and liver.See other methyltransferase structures from SGC.

Materials and Methods

AD-003

PDB:2EX4

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:56676399
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsTSEVIEDEKQFYSKAKTYWKQIPPTVDGMLGGYGHISSIDINSSRKFLQRFLREGPNKTGTSCALDCGAGIGRITKRLLLPLFREVDMVDITEDFLVQAKTYLGEEGKRVRNYFCCGLQDFTPEPDSYDVIWIQWVIGHLTDQHLAEFLRRCKGSLRPNGIIVIKDNMAQEGVILDDVDSSVCRDLDVVRRIICSAGLSLLAEERQENLPDEIYHVYSFALR
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:AD-003 was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/mL of kanamycin. Cell were grown at 37oC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.

Purification

Procedure
The crude extract was cleared by centrifugation and passing through 20-ml DE52 column equilibrated in 20 mM Tris HCl, pH 8.0, containing 500 mM NaCl and 5% glycerol. The lysate was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 20 mM Tris-HCl buffer, pH 8.0, containing 250 mM NaCl , 50 mM imidazole and 5% glycerol, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl, at flow rate 4 ml/min. Purification yield was 25.8 mg of the protein per 1L of culture.

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste was thawed and resuspended in lysis buffer (1X phosphate-buffered-saline, 0.25 M NaCl, 5 mM imidazole, 2 mM ß-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:43 mg/ml
Ligand
MassSpec:expected mass = 27282.08, measured mass = 27151.2283
Crystallization:Purified AD-003 was was complexed with S-adenosyl-L-homocysteine (SAH) (Sigma) at 1:5 molar ratio of protein:SAH and crystallized using the hanging drop vapor diffusion method at 20 °C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution containing 18% PEG3350, 0.2 M KCl, 0.1 M glycine, pH 9.5.
NMR Spectroscopy:
Data Collection:
Data Processing: