Maf domain of human N-acetylserotonin O-methyltransferase-like protein

Target ID:

ASMTL

Aliases:

ASMTLXASMTLYASTML

Deposition Date:

Friday 16th March 2007

Families:

Transferases

Related Diseases:

NeurobiologyCancer

Structure type:

apo

Download Coordinates:

2P5X

Authors:

J.MIN,H.WU,L.DOMBROVSKI,P.LOPPNAU,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

2P5X

tabs

Structure Details

Human N-acetylserotonin O-methyltransferase-like protein (ASMTL) contains two domains. The C-terminal part of the protein shows significant homology to the ASMT (N-acetylserotonin O-methyltransferase). ASMT is an enzyme catalyzing the last step in the synthesis of melatonin, a hormone that depresses body temperature and facilitates sleep onset by inducing the circadian rhythm. It is exclusively expressed in brain, retina, and pineal gland. Besides the homology of ASMTL to ASMT in its C-terminal part, the N-terminal part of ASMTL is similar to the entire length of the multicopy associated filamentation (maf) protein of Bacillus subtilis and to orfE of Escherichia coli, which are involved in the regulation of cell division and growth. The bipartite structure of ASMTL suggested that it is the product of a gene fusion event, during which 2 different full-length genes are joined to form a single gene.

Maf protein has been implicated in inhibition of septum formation in eukaryotes, bacteria and archaea. It has been predicted to be a nucleotide- or nucleic acid-binding protein with structural similarity to the hypoxanthine/xanthine NTP pyrophosphatase Ham1 from Methanococcus jannaschii, RNase H from Escherichia coli, and some other nucleotide or RNA-binding proteins. We have solved the crystal structure of Maf domain of human ASMTL protein at 2.0 Å resolution.

Materials and Methods

ASMTL

PDB:2P5X

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_004183
Entry Clone Source:
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:MGSSHHHHHHSSGLVPRGSLLHKRVVLASASPRRQEILSNAGLRFEVVPSKFKEKLDKASFATPYGYAMETAKQKALEVANRLYQKDLRAPDVVIGADTIVTVGGLILEKPVDKQDAYRMLSRLSGREHSVFTGVAIVHCSSKDHQLDTRVSEFYEETKVKFSELSEELLWEYVHSGEPMDKAGGYGIQALGGMLVESVHGDFLNVVGFPLNHFCKQLVKLYYPPRPEDLRRSVKHDSIPAADTFEDLS
Vector:pET28a-LIC

Growth

Medium:Terrific Broth (TB) in the presence of 50 μg/ml of kanamycin
Antibiotics:
Procedure: ASMTL Maf domain was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 microG/ml of kanamycin at 37degC to an OD600 of 1.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15degC

Purification

Procedure
The crude extract was cleared by centrifugation. The clarified 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 500 mM Ammonium Acetate and 50 mM imidazole, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM Ammonium Acetate, 250 mM imidazole). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 500 mM Ammonium Acetate, at flow rate 4 ml/min. Purification yield was 1.2 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 degC. For the purification the cell paste was thawed and resuspended in lysis buffer (PBS, 0.25 M NaCl, 5 mM imidazol, 2 mM &szilg;-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi
Concentration:5.7 mg/ml
Ligand
MassSpec:expected MW is 27807.6 Da, measured MW is 27676.9279 Da.
Crystallization:Purified ASMTL Maf domain was crystallized using the sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing 15% PEG 3350, 0.1 M succinic acid, pH 7.0.
NMR Spectroscopy:
Data Collection:
Data Processing: