Human BCL2 antagonist/killer 1

Target ID:

BAK1A

Aliases:

BAKBAK-LIKEBAK1BCL2L7CDN1MGC117255MGC3887

Deposition Date:

Wednesday 27th December 2006

Families:

Apoptosis

Related Diseases:

CancerNeurobiology

Structure type:

apo

Download Coordinates:

2JCN

Authors:

M.MOCHE,P.STENMARK,C.ARROWSMITH,H.BERGLUND,R.BUSAM,R.COLLINS,A.EDWARDS,U.B.ERICSSON,S.FLODIN,A.FLORES,S.GRASLUND,M.HAMMARSTROM,B.M.HALLBERG,L.HOLMBERG SCHIAVONE,I.JOHANSSON,T.KARLBERG,U.KOSINSKA,T.KOTENYOVA,S.LUNDGREN,M.E.NILSSON,T.NYMAN,D.OGG,C.PERSSON,J.SAGEMARK,M.SUNDSTROM,J.UPPENBERG,M.UPSTEN,A.G.THORSELL,S.VAN DEN BERG,J.WEIGELT,P.NORDLUND,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Stockholm

ICM Datapack:

ICM Datapack

2JCN

tabs

Structure Details

BAK1
BCL2 antagonist/killer 1 (BAK1) is, together with BAX, an ultimate death effector of apoptosis trigging cues. It is localized to the cytoplasmic side of the mitochondrial membrane, tethered by a membrane-bound C-terminal helix, and is held inactive by forming heterodimeric complexes with the antiapoptotic BCL2 family member MCL1 [1]. Upon release from MCL1, BAK1oligomerizes and permeabilizes the mitochondrial membrane, with cytochrome c release into the cytoplasm as a result [2]. Cytochrome c in turn triggers caspase activation with demounting of the cell as a result [3]. Recent results show that the tumour suppressor p53 interacts directly with BAK1 at the mitochondrial membrane [4], leading to oligomerization and release of cytochrome c.

The 1.8Ã… BAK1 structure reveals a fold most similar to that of the related BCL2-family member Bcl2-XL. Interestingly, one of the crystallographic interactions is reminiscent of interactions made between Bcl2 family members and the pro-apoptotic BH3 only proteins. This mode of interaction could also be the basis for the BAK1 oligomer interaction as well as the Bak1 â€“ Bcl2 interactions.

Materials and Methods

BAK1A

PDB:2JCN

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC004431
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*s(m).
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqSMSASEEQVAQDTEEVFRSYVFYRHQQEQEAEGVAAPADPEMVTLPLQPSSTMGQVGRQLAIIGDDINRRYDSEFQTMLQHLQPTAENAYEYFTKIATSLFESGINWGRVVALLGFGYRLALHVYQHGLTGFLGQVTRFVVDFMLHHCIARWIAQRGGWVAALNLGNGPILN
Vector:pNIC28-Bsa4

Growth

Medium:TB, supplemented with 8 g/L glycerol
Antibiotics:
Procedure:Cells (BL21(DE3)) from frozen glycerol stocks were grown in 2x20 mL of Terrific Broth, supplemented with 8 g/L glycerol, 100 µg/mL kanamycin, at 37°C over night. The following morning, 2x20 mL of the over night cultures inoculated 2x1500 mL Terrific Broth with 8 g/L glycerol , 50 µg/mL kanamycin, and 100 µL BREOX (antifoam). Cultivation was performed in glass flasks in the Large Scale Expression System (LEX). Cells were grown at 37°C until an OD600 nm of 2 was reached. The cultivations were down-tempered to 18 °C for 1h. Expression of target protein was induced by addition of 0.5 mM IPTG and was allowed to continue over night at 18 °C.

Purification

Procedure
Columns:
HiTrap Chelating 1 mL (IMAC) and HiTrap IMAC 1mL
HiLoadÂ 16/60 Superdex 75 Prep Grade (Gel filtration).

The purification was conductive manually using a peristatic pump. The duplicates were mixed and divided into two so the same amount of lysate was loaded onto the two columns (HiTrap Chelating 1mL and HiTrap IMAC 1mL). An affinity blank run was first done on the columns to avoid Ni-leakage (5 CV H2O, 5 CV W1, 5 CV E, 10 CV W1). The sample was loaded onto the columns and washed with Wash 1 buffer (5-10 CV), and Wash 2 buffer (20 CV), and eluted in elution buffer (about 18 CV). Flowrate during the runs, 1 mL/min. The protein was collected in 2 mL fractions and was detected and evaluated on SDS-PAGE. The samples were pooled and loaded onto the Gel filtration column, 16/60 Superdex 75 Prep Grade. The gelfiltration chromatogram showed one major protein peak that mainly consisted of BAK1A-c005 as shown by SDS-PAGE analysis. The protein was TEV cleaved for 4 hours and the sample was then run over a Ni-HisTrap column. The flowthrough was concentrated to 40.6 mg/mL in 0.7 mL, stored at -80ºC.

Extraction

Procedure
Cells were harvested by centrifugation (10 min, 4400 ×g) and pellets were resuspended in 50mM Sodium-Phosphate pH 7.5, 500mM NaCl, 10% glycerol, 0.5 mM TCEP (IMAC Bind/Wash1 Buffer) supplemented with one tablet of Complete EDTA-free protease inhibitor tablet and freezed at -80 ºC. The cells were quickly thawed in warm water and 4ul (1000 U) of Benzonase was added. Cells were disrupted by sonication (4s on 4s off 3 min 80% amplitude) and the lysate was centrifuged for 20 minutes at 49 000×g. The supernatant was decanted and filtered through 0.45µm filter prior to loading onto the columns, HiTrap Chelating 1mL, HiTrap IMAC 1mL and Sephadex75 for furher purification.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals was obtained by mixing 0.1+0.1 microL drops of protein solution with a well solution containing 20% (w/v) peg3350 and 0.2M sodium sulfate and were then frozen using a cryo solution containing 22% (w/v) peg3350, 20% glycerol, 0.2M sodium sulfate, 2mM TCEP, 0.3M sodium chloride, and 20mM HEPES pH 7.5. Derivative crystals were obtained by soaking experiments using saturated amounts of KAu(CN)2 and K2Cl4Pt in cryo solution for 10 min prior to freezing.
NMR Spectroscopy:
Data Collection:
Data Processing:The datasets were processed in XDS and scaled individually scaled in XSCALE before submission to XPREP for further scaling against each other. An anomalous signal was seen for both derivatives however the signal were stronger for Pt than for Au. The native data and the two derivatives were then submitted as a MIRAS job for SOLVE and RESOLVE that automatically solved and built a poly-glycine model of BAK1 with several helices. This crude model was then submitted to ARP/WARP that managed to complete 90% of all atoms using the 1.8Å dataset only. Final cycles of model building were performed in COOT using REFMAC as the refinement program.

References

Cuconati, A., Mukherjee, C., Perez, D. & White, E. (2003). DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells. Genes & Development 17, 2922-32.
Wei, M. C., Lindsten, T., Mootha, V. K., Weiler, S., Gross, A., Ashiya, M., Thompson, C. B. & Korsmeyer, S. J. (2000). tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes & Development 14, 2060-71.
Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S. & Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.[see comment]. Cell 91, 479-89.
Leu, J. I., Dumont, P., Hafey, M., Murphy, M. E. & George, D. L. (2004). Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex.[see comment]. Nature Cell Biology 6, 443-50.