Human cystathionine beta-synthase (Delta 516-525) at 2.0Ã… resolution

Target ID:

CBSA

Deposition Date:

Wednesday 29th January 2014

Families:

Nucleotide metabolism

Structure type:

protein-ligand

Download Coordinates:

4COO

Authors:

T.J.McCorvie,J.Kopec,M.Vollamar,C.Strain-Damerell,S.Bushell,A.Bradley,C.Tallant,W.Kiyani,D.S.Froese, E.S.Carpenter,N.Burgess-Brown, F. von Delft,C.Arrowsmith,A.Edwards,C.Bountra,W.W.Yue

Site:

Oxford

4COO

tabs

Structure Details

Cystathionine beta-synthase (CBS) plays an important role in the methionine and sulfur metabolism and is the only known protein to contain heme and the cofactor pyridoxal 5'-phosphate. The enzyme's canonical role is the production of cystathionine by the condensation of serine and homocystine (Hcy), which can then be metabolised to cysteine or glutathionine [1]. The enzyme is also a major contributor of hydrogen disulfide, a 'gasotransmitter' with roles in the cardiovascular and nervous system [2].
CBS enzyme deficiency (OMIM 236200) is a recessive disorder of methionine metabolism, affecting the conversion of Hcy to cystathionine. This deficiency is characterized by elevated plasma homocysteine level, with a worldwide frequency ~ 1:334,000 individuals, reaching higher incidence of 1:8,000 in certain populations. To date more than 150 disease alleles, predominantly missense mutations, have been identified on the cbs gene, with the most prevalent disease allele I278T accounting for ~ 25% of disease patients. Clinical presentations, likely due to accumulation of Hcy in various tissues, are associated with abnormalities in the eye, skeleton, vascular system and central nervous system. The major morbidity is due to thromboembolic events. Cognitive delays, seizures and neuropsychiatric symptoms can occur. A subset of patients (< 50%) respond to pharmacologic doses of pyridoxine, a precursor of PLP, resulting in reduction of Hcy, although the biochemical basis of this treatment is not clear. Patients who do not respond to pyridoxine treatment are managed with a methionine depleted diet and supplemental cystathionine. Betaine, a remethylating agent can reduce the plasma Hcy levels. Patients may be detected by newborn screening [3].
CBS adopts a modular structure with three domains (a haem binding domain, a catalytic domain and a regulatory/Bateman domain) and is tetrameric. The activity of catalytic domain is regulated by the Bateman domain, which increases activity by binding to its cognate activator, S-adenosyl-L-methionine (SAM). Until recently only the dimeric catalytic domain has been crystallized thus there is a lack in structural understanding of this regulation [4]. It has been shown that protein misfolding is the prevalent mechanism of CBS deficiency and that a pharmacological chaperone treatment has been proposed akin to phenylketonuria. Lines of evidence have also suggested targeting the Bateman domain thus not hampering the enzyme's activity. Here we report the three-domain structure of human CBS engineered with a loop deletion (delta 516-525), at 2.0 Å resolution.

Materials and Methods

CBS

PDB:4COO

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:3028099
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal, TEV protease cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:"MHHHHHHSSGVDLGTENLYFQSMPSETPQAEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKEPLWIRPDAPSRCTWQLGRPASESPHHHTAPAKSPKILPDILKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKDRISLRMIEDAERDGTLKPGDTIIEPTSGNTGIGLALAAAVRGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNARFDSPESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEILQQCDGKLDMLVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQTEQTTYEVEGIGYDFIPTVLDRTVVDKWFKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKAAQELQEGQRCVVILPDSVRNYMTKFLSDRWMLQKGFLKEEDLTEKKPWWWHLRVQELGLSAPLTVLPTITCGHTIEILREKGFDQAPVVDEAGVILGMVTLGNMLSSLLAGKVQPSDQVGKVIYKQFKQIRLTDTLGRLSHILEMDHFALVVHEQQRQMVFGVVTAIDLLNFVAAQERDQK"MHHHHHHSSGVDLGTENLYFQ*SM is the purification tag plus TEV protease recognition site *.
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure
Buffers Used:Binding/Lysis Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.01mM TCEP Wash Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.01mM TCEP Elution Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.01mM TCEP Gel Filtration Buffer: 10 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 0.01mM TCEPNi-NTA 80 mM Buffer: 50 mM Hepes (pH 7.5), 500 mM NaCl, 5% Glycerol, 80 mM Imidazole pH 7.5, 0.01mM TCEP Low Salt Buffer: 50 mM Hepes (pH 7.5), 50 mM NaCl, 5% Glycerol, 0.01mM TCEP High Salt Buffer: 50 mM Hepes (pH 7.5), 1 M NaCl, 5% Glycerol, 0.01mM TCEP Cell LysisCell pellets were dissolved in approximately 250 ml lysis buffer containg EDTA-free protease inhibitors (1 l per ml of buffer) and 0.1 mM pyridoxal 5'-phosphate. Cell pellets were thoroughly mixed using a homogenizer and then treated for 1 hour with 2 mg/ml lysozyme at 4 °C. Next the resuspended cells were broken by sonication for 10 minutes, 5 seconds on, 5 seconds of at 35 % amplitude. The insoluble cell debris was separated from the soluble fraction by centrifugation at g for 1 hour at 4 CNi-NTA IMACNi-NTA resin (1.25 ml bed volume in a gravity-flow column).The clarified soluble fraction was incubated with 3.0 ml pre-equilibriated resin slurry in 50% ratio (v/v) in binding buffer. for 1 hour at 4°C with rotation after which it spun down at g with a low brake speed. The resulting supernatant was removed and the resin pellet was resuspeneded in the remaining supernatant and was applied to a gravity column. Next the resin was washed with 2 x 20 ml of binding buffer, 2 x 20 ml wash buffer and the protein was eluted with 5 x 5 ml elution buffer. Size Exclusion Gel FiltrationSuperdex s200 16/60 Gel Filtration. Elution fractions 1 was then applied to the GF column (pre-equlibriated in GF buffer) at 1.0 ml/min. 1.0 ml fractions were collected. Hexahistidine Tag Removal and Nickel Rebind The N-terminal hexahistidine tag was cleaved by incubating overnight with TEV (20°C) at a 1:20 ratio (w/w). Cleaved protein was purified by applying the treated protein solution onto 0.5 ml of pre-equilibrated Ni-NTA. The column was then washed with 2 x 0.5 ml gel filtration buffer, 2 x 0.5 ml Ni-NTA binding buffer, 2 x 0.5 ml Ni-NTA wash buffer, 2 x 0.5 ml Ni-NTA 80 mM buffer and 2 x 0.5 ml Ni-NTA elution buffer. Cleaved fractions were isolated and combined.Anionic Exchange Chromatography Combined fractions were concentrated to 5.0 ml and then diluted in low salt buffer. This low salt solution was then applied to a pre-equilibrated Resource Q column and then the protein was eluted with a linear gradient of higher salt concentrations using the high salt buffer. Clean fractions were isolated and combined.

Extraction

Buffers
Procedure
Expression strainBL21(DE3)-R3-pRARE2A single colony each was used to inoculate 2 x 50 ml TB media supplemented with 0.002% thiamine-HCl, 0.005% pyridoxine-HCl, 0.2 mM ferric chloride, 0.6 mM ð-aminolevulinic acid, 50 ug/ml kanamycin and 50 ug/ml chloroamphenicol. These were placed in an incubator at 30 °C, 275 rpm and left to grow overnight. The next day these starter cultures were mixed and used to inoculate 6 L of TB media (10 ml starter culture used per 1 L ) grown at 30 °C, 275 rpm and supplemented with 0.002% thiamine-HCl, 0.005% pyridoxine-HCl, 0.2 mM ferric chloride, 0.6 mM ð-aminolevulinic acid and containing 50 ug/ml kanamycin. When the OD600 reached 1.8 the cells were induced with 1.0 mM IPTG and left overnight. Cell harvestCells were harvested by centrifugation at 4,500 g for 15 minutes after which the supernatant was discarded and the cell pellets were stored at -80 °C.
Concentration:To set up plates the sample was concentrated to approximatley 10.80 mg/ml using a 30 kDa mwco concentrator.
Ligand
MassSpec:Expected mass: 59584.5 Da Measured mass: 59602.6 DaDiscrepency due to water (18 Da).
Crystallization:Crystals were grown by vapour diffusion in sitting drop at 20°C. A sitting drop consisting of 50 nl protein and 100 nl well solution was equilibrated against well solution containing 18% PEG8000 -- 0.1M cacodylate pH 6.8 -- 0.2M calcium acetate
NMR Spectroscopy:
Data Collection:Resolution: 2.20 Å X-ray source: Diamond Light Source beamline IO4
Data Processing:

References

1.- Miles, E.W., and Kraus, J.P. (2004) Cystathionine beta-synthase: structure, function, regulation, and location of homocystinuria-causing mutations. J. Biol. Chem. 279, :29871-29874.

2.- Singh, S., Padovani, D., Leslie, R. A., Chiku, T., and Banerjee, R. (2009) Relative contributions of cystathionine beta-synthase and gamma- cystathionase to H2S biogenesis via alternative trans-sulfuration reactions. J. Biol. Chem. 284, 22457-22466.

3.- Kraus, J.P., Janosik, M., Kozich, V., Mandell, R., Shih, V., Sperandeo, M.P., Sebastio, G., de Franchis, R., Andria, G., Kluijtmans, L.A., Blom, H., Boers, G.H., Gordon, R.B., Kamoun, P., Tsai, M.Y., Kruger, W.D., Koch, H.G., Ohura, T., and Gaustadnes, M. (1999) Cystathionine beta-synthase mutations in homocystinuria. Hum. Mutat. 13, 362-75.

4.- Meier, M., Janosik, M., Kery, V., Kraus, J.P., and Burkhard, P. (2001) Structure of human cystathionine beta-synthase: a unique pyridoxal 5'-phosphate-dependent heme protein. Embo J. 20, 3910-3916.