Human Molybdopterin synthase catalytic subunit

Target ID:

MOCS2

Deposition Date:

Friday 30th March 2012

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

4AP8

Authors:

M.Vollmar,W.Kiyani,T.Krojer,S.Goubin,C.Allerston,D.S.Froese,F.von Delft,N.Burgess-Brown,C.Bountra,C.H.Arrowsmith,A.Edwards,W.W.Yue

Site:

Oxford

4AP8

tabs

Structure Details

Molybdenum (Mo) is a transition element that functions as an enzyme cofactor (molybdenum cofactor MoCo), when it is covalently bound to the dithiolate group of a tricyclic pterin referred to as molybdopterin (MPT) [1]. MoCo-containing enzymes participate in important redox reactions exploiting the coordination chemistry of Mo (shuttling between three oxidation states +4, +5, and +6). In mammals, the MoCo-dependent enzymes are sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase.

The MoCo cofactor is assembled in a biosynthetic pathway conserved among all living organisms, which can be divided into four reaction steps [2]: (i) GTP is converted to cyclic pyranopterin monophosphate (cPMP); (ii) Two sulphur atoms are incorporated into cPMP to form the MPT dithiolate; (iii) MPT is further processed by adenylation; and (iv) Mo metal is attached to the MPT dithiolates. Genes involved in MoCo synthesis have been identified from complementation studies on MoCo-deficient mutants, which in humans include MOCS1, MOCS2, MOCS3 and GPHN. Among them, pathogenic mutations on MOCS1, MOCS2 and GPHN are known to cause the autosomal recessive disorder of MoCo deficiency (OMIM #252150), leading to the pleiotropic loss of all MoCo-dependent enzyme activities [3]. This rare disorder is characterized by seizures, developmental delay and early childhood death.

The MOCS1 and MOCS2 genes are known to encode two overlapping open reading frames each, within a bicistronic transcript. MOCS1 encodes the MOCS1A and MOCS1B proteins involved in the initial formation of cPMP. MOCS2 encodes the small (MOCS2A) and large (MOCS2B) subunits of the heterotetrameric molybdopterin synthase complex. As a first step towards structurally characterizing the human MoCo biosynthetic pathway, we have determined the crystal structure of the molybdopterin synthase large subunit, MOCS2B.

Materials and Methods

MOCS2B (4AP8) Materials & Methods

Entry Clone Source: MGC

Entry Clone Accession: AT76-G3

Vector: pNIC-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

DNA sequence:
CGGACTCGTTCTGAAGGCGCCTCCAC
CTTTTATGACCACCTCTTTCCCAGAT
TATTCGTTTTGATGAAGCTAAAATTT
TAATCTAAAAAGAAATGCACCTCATG
GAGAATTCTTGTGAAGAACTGTGCTT
CATCTGTGGATTTCTACACCCTTGAT
CATTTGCAAACCTGTAATTATTTCGT
AAAGAGTTGTTTGCACGGAGTGACAG
GTTGAAGTATTGTATTTTGCAAAAAG
TGCTGAAATAACAGGAGTTCGTTCAG
AGACCATTTCTGTGCCTCAAGAAATA
AAAGCGTTGCAGCTGTGGAAGGAGAT
AGAAACTCGACATCCTGGATTGGCTG
ATGTTAGAAATCAGATAATATTTGCT
GTTCGTCAAGAATATGTCGAGCTTGG
AGATCAGCTCCTCGTGCTTCAGCCTG
GAGACGAAATTGCCGTTATCCCCCCC
ATTAGTGGAGGATAGTGCTTTTGAGC
CATCTAGGAAAGATATGGATGAAGTT
GAAGAGAAATCTAAAGATGTTATAAA
CTTTACTGCCGAGAAACTTTCAGTAG
ATGAAGTCTCACAGTTGGTGATTTCT
CCGCTCTGTGGTGCAATATCCCTATT
TGTAGGGACTACAAGAAATAACTTTG
AAGGGAAAAAAGTCATTAGCTTAGAA
TATGAAGCATATCTACCCATGGCGGA
AAATGAAGTCAGAAAGATTTGTAGTG
ACATTAGGCAGAAATGGCCAGTCAAA
CACATAGCAGTGTTCCATAGACTTGG
CTTGGTTCCAGTGTCAGAAGCAAGCA
TAATCATTGCTGTGTCCTCAGCCCAC
AGAGCTGCATCTCTTGAAGCTGTGAG
CTATGCCATTGATACTTTAAAAGCCA
AGGTGCCCATATGGAAAAAGGAAATA
TACGAAGAGTCATCAACTTGGAAAGG
AAACAAAGAGTGCTTTTGGGCATCCA
ACAGTTAATCACTTATGTTTTTAGAG
CATGCAATCTTAACTTTGTTAAACTA
TTATTATTGATCACATTTTGATTTTT
TTCTCTCCACATCAGGATAGTTTACT
GAAGCACAATCTCTTATACTAGTGGG
ACAAAAGGGAGAAAAAGGAAGCAAGA
TAAATGGGTATGTAGGATGAAGGGTT
ATTTAAAATGGAACTAAAGATAGAAG
GAGGACTGTAGGAAGAAATGGAATAA
TTTAAATGTGAGGAAAGATATCTGTG
GTAGACATGTCCTTCCATGACTAATT
TCTAATTGTAACTCAACACACATTGA
GGTATGGGCCCTCCTCAGTGACTTTA
ACTAGCTCAGAAACGTACTCCCCCAC
CAACCCCACCTCACCGCCCCCCATCC
CGGTTCTGGGAGAGCATTGTTATTAA
GGATGCATGACAGGAATGTTGGCAGA
ACTGGAAAGTATTAAAAAAGCATTAT
CAGACAGTCTTGATATTATACATTTT
CAGAAATATATTAAAAATAATAAACT
AAAACCCATGATTTCAAAAGTTTTAA
AAAAAAAAAAAAA

Final protein sequence (Tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^smSA
FEPSRKDMDEVEEKSKDVINFTAEKL
SVDEVSQLVISPLCGAISLFVGTTRN
NFEGKKVISLEYEAYLPMAENEVRKI
CSDIRQKWPVKHIAVFHRLGLVPVSE
ASIIIAVSSAHRAASLEAVSYAIDTL
KAKVPIWKKEIYEESSTWKGN

^ TEV protease recognition site

Tags and additions: Cleavable N-terminal His6 tag

Host: BL21(DE3)-R3-pRARE2

Growth Medium & Induction Protocol: The construct DNA was transformed into competent cells of the expression strain by a standard heat shock procedure. One colony from the transformation was used to inoculate 1.5 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture. A glycerol stock was used to inoculate 60 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to inoculate 6L of TB media (9 ml starter culture used per 1L) containing 50 µg/ml kanamycin. When the OD₆₀₀ reached approximately 1.0 the temperature was reduced to 18°C and after a further 30 minutes the cells were induced by the addition of 0.1 mM IPTG. The expression was continued overnight.

Extraction buffer, extraction method: Cell pellets were dissolved in approximately 50ml lysis buffer and broken by passing through the homogeniser (x6) at a constant pressure of 15KPa. The cell debris was pelleted at 16,000 RPM and the supernatant used for further purification.
Lysis buffer: 10 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole pH 7.4, 1 tablet per 50 ml protease inhibitor cocktail EDTA-free (Roche)

Column 1: Ni-NTA (5.0 ml volume in a gravity-flow column).

Column 1 Buffers:
Binding buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.4
Wash buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.4
Elution buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4

Column 1 Procedure: The clarified cell extract was incubated with 5.0 ml pre-equilibriated 50% Ni-NTA bead solution for 1 hour at 4°C with rotation after which it was passed through a glass column. The column was then washed with 50ml Binding Buffer (2 x 25ml) and 50 ml Wash Buffer (2 x25 ml). The protein was eluted with 50 ml of Elution Buffer in 8 x 5 ml fractions.

Column 2: Superdex s75 16/60 Gel Filtration

Column 2 Buffers:
Gel Filtration buffer: 10 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol

Column 2 Procedure: Only Elution Fractions 1-8 were pooled from the Ni-NTA column. This pool was then concentrate to 5ml and applied directly to the GF column (pre-equilibrated in GF Buffer) at 1.0 ml/min. 1.0 ml fractions were collected. During this concentration step, the protein began to crash. It was decided to halt the process and carry out a buffer exchange swaping the protein from Elution buffer into Gel filtration buffer. A second attempt to concentrate in the GF buffer was then carried out which had no effect. Concentration was nevertheless proceeded with but with careful monitoring of protein stability. The protein eluted at a volume of between 90 ml and 100 ml.

Enzymatic treatment: The N-terminal His6-tag was cleaved by incubating the protein overnight with TEV protease (20°C). Cleaved protein was purified by batch binding on 1ml pre-equilibriated 50% Ni-NTA bead solution. The column was then washed with 2 ml Gel Filtration Buffer (2x1ml) and 2 ml Binding Buffer (2x1 ml). The protein was eluted with 2 ml of Elution Buffer (2x1ml).

Gel Filtration buffer: 10 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol
Binding buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.4
Elution buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4

Mass spec characterization:
Expected: ?
Observed: ?

Concentration: To set up plates the sample was concentrated to 8.62 mg/ml using a 10 kDa mwco concentrator

Crystallization: Crystals were grown by vapour diffusion in sitting drop at 4°C. A sitting drop consisting of 50 nl protein and 100 nl well solution was equilibrated against well solution containing 0.1M HEPES pH 7.3, 0.8M potassium/sodium tartrate. Crystals were mounted in the presence of 20% (v/v) glycerol and flash-cooled in liquid nitrogen.

Data Collection:Data Collection:
Resolution: 2.78Å X-ray source: Diamond IO3

References

Rajagopalan KV, Johnson JL (1992). The pterin molybdenum cofactors. J Biol Chem 267:10199-202.
Schwarz G, Mendel RR, Ribbe MW (2009). Molybdenum cofactors, enzymes and pathways. Nature 460:839-47
Reiss J, Hahnewald R (2011). Molybdenum cofactor deficiency: Mutations in GPHN, MOCS1, and MOCS2. Hum Mutat 32:10-8.