Human S-adenosylmethionine synthetase 2, beta subunit in orthorhombic crystal form

Target ID:

MAT2BB

Aliases:

MAT-IIMAT2BMATIIbetaMGC12237Nbla02999TGR

Deposition Date:

Thursday 25th March 2010

Families:

Oxidoreductases

Related Diseases:

MetabolismCancerDrug metabolism and toxicology

Structure type:

apo

Download Coordinates:

2YDY

Authors:

W.W.YUE,N.SHAFQAT,J.R.C.MUNIZ,T.KROJER,A.C.W.PIKE,A.CHAIKUAD,C.K.ALLERSTON,O.GILEADI,F.VON DELFT,K.L.KAVANAGH, C.H.ARROWSMITH,A.M.EDWARDS,J.WEIGELT,C.BOUNTRA,U.OPPERMANN

Site:

Oxford

ICM Datapack:

ICM Datapack

2YDY

tabs

Structure Details

Methionine adenosyltransferases (MAT) are the key enzymes for synthesizing S-adenosyl-methionine (SAM), the principal methyl donor in biological methylations, in a reaction utilizing ATP and L-methionine as substrates. In mammals there are two MAT catalytic isoforms: a liver-specific subunit that exists in the tetrameric (MATI) or dimeric (MATIII) states, and a ubiquitously-expressed subunit (MATIIα) that hetero-oligomerizes with its regulatory subunit MATIIβ. MATIIβ, which interacts only with MATIIα but not MATI/III, belongs to the enzyme superfamily of short-chain dehydrogenases/reductases [1]. It regulates the kinetic properties of MATIIα by lowering the KM for its substrate methionine [2]. Therefore in comparison to MATI/III, MATIIα has higher affinity for its substrate methionine, and at the same time becomes more susceptible for feedback inhibition by its product SAM [3]. The ability of MATIIβ to modulate MATIIα activity provides an effective means in controlling the intracellular levels of SAM which are needed at different stages of cell differentiation.

MATIIα and MATIIβ have both been detected in the nucleus, supporting a chromatin-associated function. To this end, MATIIα was recently shown to be recruited to the promoter of the heme oxygenase-1 (HO-1) gene and, in concert with the MafK transcription factor, repress transcription of HO-1 in a manner dependent on the MATIIα catalytic activity [4]. This illustrates a potential role for MATIIα and MATIIβ in regulating gene expression via the biosynthesis of the SAM metabolite, a precursor for chromatin methylation. MATIIβ is also involved in cell proliferation and has been associated with cirrhosis and cancer in hepatoma cells via its interaction with MATIIα [5]. We have previously crystallized the catalytic subunits of MATI/MATIII (PDB code 2OBV) and MATIIα (PDB code 2P02). Here we present the structure of the MATIIβ regulatory subunit in binary complex with NADP at 2.80Å resolution, as well as the structure of a truncated MATIIβ construct in the apo state at 2.25Å resolution.

Materials and Methods

Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: MAT2BB-c018

GenBank GI number: gi|33519455

Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
CTTAAGAAGGAGATATACTATGAACC
GTCGCGTTCTGGTTACCGGTGCTACT
GGTCTGCTGGGTCGTGCAGTACACAA
GGAGTTTCAGCAGAATAACTGGCACG
CTGTAGGTTGCGGTTTTCGTCGTGCG
CGTCCAAAATTCGAGCAAGTAAACCT
GCTGGACTCTAACGCTGTTCACCACA
TCATCCACGATTTCCAGCCGCACGTT
ATTGTGCATTGCGCTGCTGAACGTCG
TCCTGACGTTGTAGAAAACCAGCCAG
ATGCTGCCTCCCAACTGAACGTTGAT
GCGTCTGGTAACCTGGCCAAGGAAGC
TGCTGCAGTAGGCGCATTTCTGATCT
ATATCAGCTCCGATTACGTTTTCGAC
GGTACCAACCCGCCTTATCGTGAGGA
AGACATCCCGGCTCCACTGAACCTGT
ACGGTAAGACGAAGCTGGATGGCGAA
AAAGCGGTGCTGGAAAATAACCTGGG
TGCTGCAGTTCTGCGTATCCCGATCC
TGTATGGTGAAGTCGAAAAACTGGAA
GAATCCGCGGTTACTGTTATGTTCGA
CAAGGTCCAGTTCTCCAATAAGTCTG
CAAACATGGACCATTGGCAACAGCGC
TTCCCGACTCACGTTAAAGATGTGGC
TACGGTTTGCCGCCAACTGGCAGAGA
AGCGTATGCTGGACCCGAGCATCAAG
GGTACTTTCCATTGGAGCGGCAACGA
ACAGATGACGAAATACGAAATGGCAT
GTGCGATTGCCGACGCATTCAACCTG
CCATCTTCTCACCTGCGTCCGATCAC
CGATTCCCCAGTGCTGGGTGCACAAC
GTCCACGTAATGCGCAACTGGACTGT
TCTAAACTGGAAACTCTGGGTATCGG
TCAGCGCACTCCATTTCGTATCGGTA
TTAAGGAGTCTCTGTGGCCGTTCCTG
ATCGATAAACGTTGGCGTCAGACTGT
GTTCCATGCAGAGAACCTCTACTTCC
AATCGCACCATCATCACCACCATGAT
TACAAGGATGACGACGATAAGTGAGG
ATCC

Final protein sequence (Tag sequence in lowercase):
MNRRVLVTGATGLLGRAVHKEFQQNN
WHAVGCGFRRARPKFEQVNLLDSNAV
HHIIHDFQPHVIVHCAAERRPDVVEN
QPDAASQLNVDASGNLAKEAAAVGAF
LIYISSDYVFDGTNPPYREEDIPAPL
NLYGKTKLDGEKAVLENNLGAAVLRI
PILYGEVEKLEESAVTVMFDKVQFSN
KSANMDHWQQRFPTHVKDVATVCRQL
AEKRMLDPSIKGTFHWSGNEQMTKYE
MACAIADAFNLPSSHLRPITDSPVLG
AQRPRNAQLDCSKLETLGIGQRTPFR
IGIKESLWPFLIDKRWRQTVFHaenl
yfqshhhhhhdykddddk

Tags and additions: C-terminal His-tag with TEV protease cleavage site

Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain).

Growth medium, induction protocol: 10µl of a glycerol stock was inoculated into 5ml of LB medium (supplemented with 50µg/ml Kanamycin, 34µg/ml Chloramphenicol) and cultured at 37°C o/n in a shaking incubator (275 rpm). Next evening the o/n starter culture was used to inoculate 100ml of LB medium and was grown at 37°C (200 rpm) o/n. Next morning at an OD₆₀₀ of 2.2 the culture was harvested and the cell pellet was washed twice with M9 minimal medium (Molecular Dimensions Ltd). The cells were resuspended and used to inoculate 1 liter of prewarmed minimal medium. Methionine synthesis was suppressed by addition of leucine, isoleucine and valine (dissolved as 50mg/l for each aa) and lysine, threonine, and phenylalanine (100mg/l of each aa). Selenomethionine was added to a concentration of 25mg/l, and at an OD₆₀₀ of 1.0 cells were induced by supplementation with 1 mM IPTG. Cells were grown overnight at 18°C, collected by centrifugation and stored frozen until further use.
Lysis buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5 mM Imidazole; 5% Glycerol; Complete® protease inhibitors (Roche, 1 tbl/50ml).
Extraction buffer, extraction method: Frozen pellets were thawed and resuspended in a total volume of 30-40ml of lysis buffer, and disrupted by using Avestin C-5 microfluidizer, and a supernatant containing the target protein was obtained by centrifugation at 21,000rpm for 45 minutes.

Column 1: Ni-Sepharose 6 Fast Flow

Column 1 Buffers:
Lysis buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5 mM TCEP; 5 mM Imidazole.
Wash buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5 mM TCEP; 30 mM Imidazole.
Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5 mM TCEP; 250 mM Imidazole.

Column 1 Procedure: The column was packed with 2ml of Ni-Sepharose 6 Fast Flow slurry and equilibrated with 15ml of binding buffer. The supernatant was loaded onto the column and the column was washed with 20ml of binding buffer and then 20ml of washing buffer. The protein was eluted with 10ml of elution buffer.

Column 2: Superdex S200 16/60 HiLoad (GE/Amersham)

Column 2 Buffer: 10 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5 mM TCEP.

Column 2 Procedure: The eluted protein from the Ni-affinity column was loaded on the gel filtration column in GF buffer at 1.0ml/min on an ÄKTA Purifier system. Eluted protein was collected in 1ml fractions.

Subtilisin treatmeant of MAT2B protein: Around 18mg of MAT2B protein at a concentration of 8.9mg/ml was treated with 0.6mg of subtilisin for 40 minutes at room temperature. After 40 minutes, reaction was stopped by adding 20µl of 0.5 M PMSF. Later sample was loaded on gel filtration column to remove PMSF.

Column 3: Superdex S200 16/60 HiLoad (GE Amersham)

Column 3 Buffer: 10 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5 mM TCEP.

Column 3 Procedure: The subtilisin-treated protein was loaded on the gel filtration column in GF buffer at 1.0ml/min on an ÄKTA Purifier system. Eluted protein was collected in 1ml fractions.

Mass spectrometry characterization: The truncated mass of selenomethionine labelled MAT2B (subtilisin treated) was 24675Da, as determined by ESI-TOF MS.

Protein concentration: Protein was concentrated to 16mg/ml using an Vivaspin 10k cut-off concentrator and stored at -80°C.

Crystallisation: Crystals were grown by vapour diffusion in sitting drops at 20°C. A sitting drop consisting of 100nl protein and 50nl well solution was equilibrated against well solution containing 0.1 M Hepes pH 7.5 and 1.5 M Lithium sulphate. The crystals were mounted directly from the drop using 25% glycerol as a cryoprotectant and flash-cooled in liquid nitrogen.

Data collection: Resolution: 2.20Å. Phasing: Synchrotron Diamond Light Source beamline I04, single wavelength.

References

Persson, B., et al., The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative. Chem Biol Interact, 2009. 178(1-3): p. 94-8.
Kotb, M. and A.M. Geller, Methionine adenosyltransferase: structure and function. Pharmacol Ther, 1993. 59(2): p. 125-43.
Halim, A.B., et al., Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells. J Biol Chem, 1999. 274(42): p. 29720-5.
Katoh, Y., et al., Methionine adenosyltransferase II serves as a transcriptional corepressor of Maf oncoprotein. Mol Cell, 2011. 41(5): p. 554-66.
Wang, Q., et al., Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma. World J Gastroenterol, 2008. 14(29): p. 4633-42.