Human spermine synthase in complex with spermine and 5-methylthioadenosine

Target ID:

SMS

Aliases:

MRSRSPMSYSpSSRS

Deposition Date:

Monday 4th February 2008

Families:

Transferases

Related Diseases:

Neurobiology

Structure type:

protein-ligand

Download Coordinates:

3C6M

Authors:

Min J, Wu H, Zeng H, Loppnau P, Weigelt J, Sundstrom M, Arrowsmith CH, Edwards AM, Bochkarev A, Pegg AE, Plotnikov AN

Site:

Toronto

3C6M

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Structure Details

The nearly ubiquitous polyamines (putrescine, spermidine and spermine) are mediators of cell proliferation and differentiation. They may act as ligands at multiple sites on DNA, RNA, proteins, phospholipids, and nucleotide triphosphates. In normal cells, polyamine levels are intricately controlled by biosynthetic and catabolic enzymes. The strong correlation between polyamine synthesis and cell proliferation makes the polyamine biosynthetic and catabolic enzymes attractive targets for the development of antiproliferative therapeutics.

Spermine synthase is one of the enzymes involved in polyamine metabolism. It transfers an aminopropyl group from decarboxylated S-adenosylmethionine to spermidine, leading to the formation of spermine. We have solved the crystal structure of human spermine synthase in complex with spermine and 5'-methylthioadenosine at 1.8 Å resolution. The hsSMS consists of three domains: the N-terminal domain including 7 β strands and 2 α helixes; the center β strand domain including 4 β strands; the C-terminal catalytic domain, which has a canonical methyltransferase fold. The N-terminal domain structure is similar to s-adenosylmethionine decarboxylase proenzyme (PDB: 1TLU). The structure provides us important information for understanding of catalytic mechanism of polyamine biosynthesis. It will also facilitate the selection of effective therapeutic agents for the treatment of a particular type of malignancy.

Materials and Methods

SMS

PDB:3C6M

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:21264341
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 (Stratagene).

Construct

Prelude:
Sequence:gsRHSTLDFMLGAKADGETILKGLQSIFQEQGMAESVHTWQDHGYLATYTNKNGSFANLRIYPHGLVLLDLQSYDGDAQGKEEIDSILNKVEERMKELSQDSTGRVKRLPPIVRGGAIDRYWPTADGRLVEYDIDEVVYDEDSPYQNIKILHSKQFGNILILSGDVNLAESDLAYTRAIMGSGKEDYTGKDVLILGGGDGGILCEIVKLKPKMVTMVEIDQMVIDGCKKYMRKTCGDVLDNLKGDCYQVLIEDCIPVLKRYAKEGREFDYVINDLTAVPISTSPEEDSTWEFLRLILDLSMKVLKQDGKYFTQGNCVNLTEALSLYEEQLGRLYCPVEFSKEIVCVPSYLELWVFYTVWKKAKP
Vector:pET28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:SMS was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 μg/ml of kanamycin. Cells were grown at 37oC to 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 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 and 50 mM imidazole, 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. Thrombin (Sigma) was added to combined fractions containing SMS and incubated overnight at 4oC. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30Q column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM Tris-HCl, pH 8.0, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 2.3 mg of the protein per 1L of culture.
Enzymatic treatment: Thrombin

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 purification the cell paste was thawed and resuspended in lysis buffer (phosphate-buffered saline, pH 7.4, 0.25 M NaCl, 5 mM imidazol, 2 mM β-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:21.8 mg/ml
Ligand
MassSpec:expected MW = 41067.95 Da, measured MW = 41055.637 Da.
Crystallization:Purified was complexed with spermine and 5Â-methylthioadenosine (protein: SPM:MTA ratio at 1:5:5) and crystallized using the hanging drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing18 % PEG 20000, 0.1 M NaCl, 0.1 M BisTris, pH 6.5.
NMR Spectroscopy:
Data Collection:
Data Processing: