Human cystathionine-beta-synthase

Target ID:

CBSA

Deposition Date:

Thursday 31st July 2014

Families:

Miscellaneous

Related Diseases:

Metabolism

Structure type:

protein-ligand

Download Coordinates:

4UUU

Authors:

J.Kopec, T.J.McCorvie, F.Fitzpatrick, C.Strain-Damerell, D.S. Froese, C. Tallant, N.Burgess-Brown, C.Arrowsmith, A.Edwards, C.Bountra, W.W.Yue

Site:

Oxford

4UUU

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 crystal structure of SAM-bound CBS regulatory domain engineered with a loop deletion (delta 516-525), at 1.7 Å resolution.

Materials and Methods

CBS

PDB:4UUU

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

Growth

Medium:
Antibiotics:
Procedure:

Purification

Procedure
Cell LysisCell pellets were dissolved in approximately 60 ml lysis buffer containg EDTA-free protease inhibitors (diluted 1:1000). The resuspended cell pellets were then treated for 1 hour with 2 mg/ml lysozyme at 4 °C and then broken by homogenization. The insoluble cell debris was separated from the soluble fraction by centrifugation. Talon IMACThe clarified soluble fraction was incubated with 3.0 ml pre-equilibriated Talon resin slurry in 50% ratio (v/v) in binding buffer. for 1 hour at 4°C with rotation. After it was centrigued with a low brake speed to pellet the resin. 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 1 x 10 ml of binding buffer, 5 x 5 ml wash buffer and the protein was eluted with 6 x 3 ml and 2 x 5 ml elution buffer. Size Exclusion Gel FiltrationSuperdex s75 16/60 Gel Filtration. Pooled elution fractions were then applied to the GF column (pre-equlibriated in GF buffer) at 1.0 ml/min. 1.0 ml fractions were collected. 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

Procedure
BL21(DE3)-R3-pRARE2A single colony each was used to inoculate 2 x 50 ml TB media with 50 ug/ml kanamycin and 50 ug/ml chloroamphenicol. These were placed in an incubator at 37 °C, 180 rpm and left to grow overnight. The next day these starter cultures were mixed and used to inoculate 3 L of TB media (10 ml starter culture used per 1 L ) grown at 37 °C, 180 rpm and supplemented with 50 ug/ml kanamycin and 50 ug/ml chloroamphenicol.. When the OD600 reached 1.8 the cells were induced with 0.1 mM IPTG AT 18 °C 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 5 mg/ml using a 10 kDa MWCO concentrator.
Ligand
MassSpec:Expected mass: 17425.2 Da Measured mass: 17426.17 Da
Crystallization:Crystals were grown by vapour diffusion in sitting drop at 20°C. A sitting drop consisting of 100 nl protein with 1 mM S-adenosyl-L-methionine and 50 nl well solution was equilibrated against well solution containing 36% PEG550MME -- 0.1M tris pH 7.5 -- 0.2M calcium chloride.
NMR Spectroscopy:
Data Collection:Resolution: 1.9 Å X-ray source: Diamond Light Source beamline IO2
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., Janosík, 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.