Human xenobiotic/medium-chain fatty acid:CoA ligase (L64P mutation) in complex with AMP

Target ID:

LOC148158A

Aliases:

ACSM2ACSM2BHXMAHYST1046

Deposition Date:

Wednesday 30th July 2008

Families:

Miscellaneous

Related Diseases:

MetabolismDrug metabolism and toxicology

Structure type:

protein-ligand

Download Coordinates:

2VZE

Authors:

W.W.YUE,G.T.KOCHAN,E.S.PILKA,C.BHATIA,F.VON DELFT,C.H.ARROWSMITH,A.M.EDWARDS,M.WIKSTROM,C.BOUNTRA,U.OPPERMANN

Site:

Oxford

ICM Datapack:

ICM Datapack

2VZE

tabs

Structure Details

The adenylate forming superfamily of enzymes are characterized by utilizing ATP and an acyl- or aryl- carboxylate to form an adenylate reaction intermediate. Within this superfamily, acyl- and aryl-CoA synthetases form one of three subfamilies. Members of this group further catalyze the thioester formation of the acyl/aryl adenylate intermediate by using CoA or ACP. We now solved the first mammalian structure of this class of enzymes.

Within the human genome several genes with putative acyl/aryl CoA ligase activity have been identified, with distinct specificities towards their endogenous fatty acid substrates. At least one member (LOC148158), which is a medium-chain acyl-CoA synthase, has also considerable activity towards carboxylic acid group containing xenobiotics such as for example benzoate or ibuprofen.

LOC148158 is highly similar (>95%) to human type 2 medium-chain acyl CoA synthase (MACS2), where a Leu513Ser polymorphism has been associated as risk factor for the development of the metabolic syndrome in caucasians.

We have determined the structure of human LOC148158 in complex with AMP, the by-product of the thioester-forming step of the catalytic reaction, revealing significant different binding modes for AMP and ATP.

Materials and Methods

Vector: pFB-LIC-Bse. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: N-terminal, TEV cleavable hexahistidine tag. Tag sequence: mhhhhhhssgvdlgtenlyfq(*)sm

Host:Trichoplusia Ni (High five)

Sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq*smSLQWG
HQEVPAKFNFASDVLDHWADMEKAGKRPP
SPALWWVNGKGKELMWNFRELSENSQQAA
NVLSGACGLQRGDRVAVVLPRVPEWWLVI
LGCIRAGLIFMPGTIQMKSTDILYRLQMS
KAKAIVAGDEVIQEVDTVASECPSLRIKL
LVSEKSCDGWLNFKKLLNEASTTHHCVET
GSQEASAIYFTSGTSGLPKMAEHSYSSLG
LKAKMDAGWTGLQASDIMWTISDTGWILN
ILCSLMEPWALGACTFVHLLPKFDPLVIL
KTLSSYPIKSMMGAPIVYRMLLQQDLSSY
KFPHLQNCVTVGESLLPETLENWRAQTGL
DIRESYGQTETGLTCMVSKTMKIKPGYMG
TAASCYDVQIIDDKGNVLPPGTEGDIGIR
VKPIRPIGIFSGYVDNPDKTAANIRGDFW
LLGDRGIKDEDGYFQFMGRADDIINSSGY
RIGPSEVENALMEHPAVVETAVISSPDPV
RGEVVKAFVVLASQFLSHDPEQLTKELQQ
HVKSVTAPYKYPRKIEFVLNLPKTVTGKI
QRAKLRDKEWKMSGKARAQ

Expression: High five cells were grown in Sf900II medium supplemented with 1% FCS at 27°C. Cells were infected at a density of 2x106/ml with recombinant baculovirus (virus stock P2; 1ml of virus stock/1l of cell culture). 72 hours post-infection the cultures were collected and centrifuged for 30min at 2000rpm. The cell pellet was resuspended in cold PBS and centrifugation was repeated. The pellet was resuspended in lysis buffer (50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml)) and frozen at -80°C until purification.

Extraction: Lysis buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml).The thawed cellular lysates were supplemented with Igepal CA 630 (Fluka) at a final concentration of 0.05%, and benzonase (Novagen) (25u/10ml of lysate). Cells were broken using a Dounce homogeniser, followed by centrifugation for 45 min at 20.000rpm at 4°C.

Column 1: Ni-affinity, Ni-Sepharose - (GE Healthcare) purification in batch

Column 2: Superdex 200Column , HiPrep 16/60 (Amersham)

Column 3: Ion exchange -5ml HiTrap Q Sepharose

Buffers: Start buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 10mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 250mM Imidazole, 0.5mM TCEP; GF buffer: 10mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 0.5mM TCEP; IEX buffers: Buffer A: 50mM Tris pH 9.0, Buffer B: 50mM Tris pH 9.0, 1M NaCl.

Procedure: The cell extract was incubated with Ni-Sepharose (0.250ul of resin/lysate from 1l of culture) during 1h at 4°C with gentle rotation. The resin was centrifuged at 1000g for 5min at 4°C, and washed 4x with 50ml of washing buffer. Resin was loaded on the gravity column and protein was eluted in 4 elution fractions (5ml each). Protein fractions were analysed by SDS-PAGE. Target protein containing fractions were concentrated using Amicon Ultra-15 concentrators with 10kDa cut-off, and purified on a gel filtration column (Superdex 200 ) on an Akta Purifier System. LOC148158 containing fractions were diluted with IEX buffer A to a concentration of NaCl of 50 mM. The protein was loaded onto a HiTrap Q Sepharose column, and eluted with a NaCl gradient (50-300mM). Fractions containing protein were analysed by SDS-PAGE.

Concentration and buffer exchange: Using Amicon Ultra-15 concentrators with 10kDa cutoff, the sample was concentrated to 20mg/ml. Concentrations were determined from the absorbance at 280 nm using a NanoDrop spectrophotometer.

Mass spec characterization: The calculated mass of the construct was 63260Da, and the observed mass (ESI-MS) was 63173Da, compatible with an N-terminal methionine deletion and subsequent acetylation at the N-teminus of the protein.

Crystallization: Crystals were grown by vapor diffusion at 20°C in 150nl sitting drops. AMP was added to a final concentration of 5mM prior to crystallisation. The drops were prepared by mixing 100nl of protein solution and 50nl of precipitant consisting of 0.2M (NH₄)(ac); 0.1M TRIS pH 8.5; 25% PEG 335. Crystals were flash-cooled in liquid nitrogen with 25 % glycerol as cryoprotectant.

Data Collection: Resolution: 2.45Å. X-ray source: Swiss Light source (SLS), beamline X-10.