Human BTB and CNC homology 1

Target ID:

BACH1A

Deposition Date:

Tuesday 26th September 2006

Families:

BTB-Kelch

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2IHC

Authors:

A.L.AMOS,A.P.TURNBULL,G.BUNKOCZI,E.PAPAGRIGORIOU,F.GORREC,C.UMEANO,A.BULLOCK,F.VON DELFT,J.WEIGELT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,S.KNAPP,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2IHC

tabs

Structure Details

BACH1 belongs to the small basic region leucine zipper (bZIP) family Maf of transcriptional activators/repressors. BACH1 contains a BTB (broad complex, tramtrack, bric-a-brac) domain, also known as a POZ (poxvirus and zinc finger) domain. The BTB/POZ domains facilitate protein-protein interactions and formation of homo-and/or heterooligomers. BACH1 is localized in the cytoplasm whereas a shorter isoform (BACH1t) accumulates in the nucleus. Inhibition of heme synthesis enhances nuclear accumulation of BACH1, whereas treatment of cells with hemin results in nuclear exclusion.

BACH1 activity is regulated by oxidation and BACH1 may function as a transcriptional activator or repressor depending on the cellular context. The protein regulates antioxidant enzymes, in particular hemeoxygenase 1 (HO-1), which is an antioxidant defense enzyme that converts toxic heme into antioxidants and is essential for higher eukaryotes in order to cope with various aspects of cellular stress and to regulate cellular iron metabolism.

BACH1 heterodimerises with the basic region leucine zipper (bZip) protein MafK. Complex formation is mediated by its BTB domains and generates multimeric and multivalent DNA binding complexes. In addition, BACH1 dimerizes with its shorter isoform BACH1t resulting in nuclear import of BACH1.

BACH1 knock-out mice are viable and fertile and appear grossly normal in size and morphology. However, BACH1 -/- mice have constitutive high expression of HO-1 in various tissues under normal physiological conditions (uncoupled from stress-responsive control) and are resistant to pro-atherosclerotic and ischemic stresses. In addition, smooth muscle cell proliferation is reduced in BACH1 -/- mice.

Transgenic mice expressing human BACH1 under the control of GATA-1 show significant thrombocytopenia associated with impaired maturation of megakaryocytes and reduced proplatelet formation. The modal ploidy class of megakaryocytes in these mice was 2N, indicating impairment of endomitosis. In addition these transgenic mice develop myelofibrosis and show a down-regulated transcription of the p45 target genes.

BACH1 plays critical roles in the regulation of macrophages and SMC functions and pathogenesis of atherosclerosis and is a potential target for antiatherosclerotic /antiinflammatory therapy. Aberrant BACH1 expression has been reported in brains of fetuses with Down syndrome and patients with Down syndrome and Alzheimer's disease.

Materials and Methods

BACH1

PDB:2IHC

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|45827690
Entry Clone Source:MGC
SGC Clone Accession:
Tag: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmSVFAYE SSVHSTNVLLSLNDQRKKDVLCDVTIFVE GQRFRAHRSVLAACSSYFHSRIVGQADGE LNITLPEEVTVKGFEPLIQFAYTAKLILS KENVDEVCKCVEFLSVHNIEESCFQFLKF
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Starter cultures ( 10 ml LB, 50 µg/ml kanamycin) were inoculated with a single colony and grown overnight. LB media (1L) was inoculated with 5 ml of culture and grown at 37°C, 170 rpm until OD600 was 0.4. The flask was then transferred to an 18°C incubator and protein expression induced with 0.1 mM IPTG (final concentration) at an OD600 of approximately 0.5. Cells were harvested the following morning by centrifugation (10min, 6500 rpm, 4°C). The pellet was resuspended in 25 ml binding buffer and frozen at -20°C.

Purification

Procedure
Columns 1 & 2 : DE52 anion exchange resin/ IMAC Sepharose 6 Fast Flow resin, placed in series.
Gravity feed chromatography: DE52 anion exchange resin was prepared by suspending 10 g in 100 ml of 2.5 M NaCl. The resulting gel slurry was packed in a column and allowed to settle prior to equilibrating in approximately 50 ml of Binding Buffer. Nickel charged IMAC sepharose beads were prepared by packing 4 mls of gel slurry (approximately 50% beads in 30% EtOH) in a small column and equilibrating in approximately 10 ml of Binding Buffer. Both columns were then placed in series and the protein containing supernatant passed through under gravity. This was followed by washing using 50 ml of Binding Buffer followed by 50 ml of Wash Buffer. Elution of the protein proceeded using a stepwise gradient of elution buffer: 5 mls x 30 mM imidazole, 5 ml x 50 mM imidazole and 10 ml x 250 mM imidazole.

Enzymatic treatment: Protein containing fractions were pooled and the His-tag cleaved using TEV protease (reaction proceeded overnight at 4°C).

Column 3: Gel Filtration, HiLoad 16/60 Superdex 75 prep grade column run on an AKTA-prime system.
The protein solution was concentrated using a Vivaspin 20 ultrafiltration device with a 5 kDa MWCO to about 10 ml. A pre-equilibrated gel filtration column was injected with 5 ml of the solution and two runs undertaken at a flow rate of 0.8 ml/min.

Concentration: Using Vivaspin 5 ultrafiltration spin columns with a 5 kDa MWCO the protein was dialysed against 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.5 mM TCEP and concentrated to 5 mg/ml approximated using UV absorbance at 280 nm and a predicted extinction coefficient of 4720.

Extraction

Procedure
The cell pellet was defrosted and AEBSF plus CHAPS were added to a final concentration of 1 mM and 0.5% (w/v) respectively. Cells were lysed by sonication, 10 s on, 2 s off at 40% amplitude for 8 min. The lysate was cleared by centrifugation for 45 minutes at 21,500 rpm, 4°C.
Concentration:
Ligand
MassSpec:Following TEV treatment of the protein mass spec confirmed the correct mass of 14,042 Da expected for this construct.
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/ml by mixing 750nl of the concentrated protein with 750nl of a well solution containing 20% PEG3350, 0.2M MgCl2, 0.1M Tris pH 8.5. Crystals appeared after several days at 20°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution supplemented with an additional 15% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line X10 ( l =0.979 Å) to 2.45Å
Data Processing:

References

Ferrando-Miguel, R., Cheon, M. S., Yang, J. W., and Lubec, G. (2003). Overexpression of transcription factor BACH1 in fetal Down syndrome brain. J Neural Transm Suppl, 193-205.
Kanezaki, R., Toki, T., Yokoyama, M., Yomogida, K., Sugiyama, K., Yamamoto, M., Igarashi, K., and Ito, E. (2001). Transcription factor BACH1 is recruited to the nucleus by its novel alternative spliced isoform. J Biol Chem 276 , 7278-7284.
Oyake, T., Itoh, K., Motohashi, H., Hayashi, N., Hoshino, H., Nishizawa, M., Yamamoto, M., and Igarashi, K. (1996). Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site. Mol Cell Biol 16 , 6083-6095.
Shim, K. S., Rosner, M., Freilinger, A., Lubec, G., and Hengstschlager, M. (2006). Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells. Exp Cell Res 312, 2264-2278.
Toki, T., Katsuoka, F., Kanezaki, R., Xu, G., Kurotaki, H., Sun, J., Kamio, T., Watanabe, S., Tandai, S., Terui, K. , et al. (2005). Transgenic expression of BACH1 transcription factor results in megakaryocytic impairment. Blood 105 , 3100-3108.