Human BTB domain protein BDPL

Target ID:

BTBD6A

Aliases:

BDPLBTBD6

Deposition Date:

Friday 21st December 2007

Families:

BTB-KelchUbiquitin Related BiologyUbiquitylation - E3 Ligases

Structure type:

apo

Download Coordinates:

2VKP

Authors:

C.D.O.COOPER,A.C.W.PIKE,E.SALAH,P.FILIPPAKOPOULOS,G.BUNKOCZI,J.M.ELKINS,F.VON DELFT,O.GILEADI,A.EDWARDS,J.WEIGELT,C.H.ARROWSMITH,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2VKP

tabs

Structure Details

The BTB domain (named after the Drosophila transcription factors Bric-a-brac, Tramtrack, and Broad) is a protein-protein interaction motif found in all eukaryotes. Sequence conservation of this motif is highly variable and widely distributed in diverse proteins. BTB domains may serve as oligomerization domains or function as binding interfaces with other proteins.

BTBD6 belongs to a BTB subfamily with a BTB-BACK-PHR domain architecture. Other members of this family including BTBD1 and BTBD2 have been shown to interact with topoisomerase 1 through the PHR domain (pfam PF08005). This C-terminal region of ~170 amino acids is predicted to represent a novel type of β-propeller structure and has sequence similarity with human protein associated with myc (PAM; NCBI accession number AAC39928), Drosophila highwire (AAF76150) and C. elegans regulator of presynaptic morphology (RPM-1; NP_505267.1).

BTBD6 expression has been found in the human lens giving rise to the alternative name “Lens BTB domain protein”. The identified ESTs match a partial sequence previously described as “glucocorticoid receptor AF-1 coactivator-1” (AF173358) which was isolated from placenta by GST pull down.

In order to gain insight into the structure and function of BTBD6 we determined the structure of its N-terminal BTB domain at 1.6 Å resolution. This structure based on the primary swiss prot entry (Q96KE9) reveals the highly unusual deletion of the b1 and a1 elements of the dimerization interface. These may be restored, and therefore have a potential regulatory function, in an alternative initiation site variant (gi: 119602320) which includes a further N-terminal proline rich region also present in related BTB domain proteins.

Materials and Methods

BTBD6

PDB:2VKP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:4824525
Entry Clone Source:MGC
SGC Clone Accession:
Tag: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence: mhhhhhhssgvdlgtenlyfq*sMFNNELMADVHFVVGPPGATRTVPAHKYVLAVGSSVFYAMFYGDLAEVKSEIHIPDVEPAAFLILLKYMYSDEIDLEADTVLATLYAAKKYIVPALAKACVNFLETSL
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:The expression plasmid was transformed into the host strain and plated on LB-agar containing 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. Several colonies were combined to inoculate a 1-ml culture in TB (+ 50 µg/ml kanamycin, 35 µg/ml chloramphenicol). The culture was grown overnight, glycerol was added to 15% v/v (from a 60% stock), and the resulting glycerol stock was frozen at -80°C in 100 µl aliquots. A loopful of cells from the glycerol stock was inoculated into 6x 10-ml of LB medium containing 100 µg/ml kanamycin and 35 µg/ml chloramphenicol and grown overnight at 37°C. Cultures were harvested by centrifugation and washed twice with M9 minimal medium and resuspended in 10 ml M9 minimal medium. 6x 1L M9 minimal medium (containing 0.4% glucose, 2mM MgSO4, 0.1mM CaCl2, 50 µg/ml kanamycin) were each inoculated with 10 ml resuspended cells and grown in 2.5L UltraYield baffled flasks until OD600 of 0.80. Selenomethioine was added to 25mg/L along with leucine, isoleucine and valine to 50mg/L and lysine, threonine, and phenylalanine to 100mg/L (all amino acids dissolved in 0.2M HEPES pH 7.5). Cultures were grown for a further 1.5 hours until OD600 of 1.2 and then cooled to 18°C for 1 hour. Additional selenomethioine was added (final total concentration of 75mg/L) and IPTG was added to 0.1 mM, and growth continued at 18°C overnight. The cells were collected by centrifugation then the pellets were scraped out and transferred to 50-ml Falcon tubes and frozen at -80°C.

Purification

Procedure

Column 1 : Ni-affinity, HisTrap Crude FF, 5 ml (GE Healthcare)

Solutions: Affinity buffer: 50 mM HEPES buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% Glycerol, 0.5 mM TCEP. Wash buffer: 50 mM HEPES buffer, pH 7.5, 500 mM NaCl, 30 mM imidazole, 5% Glycerol, 0.5 mM TCEP. Elution buffer: 50 mM HEPES buffer, pH 7.5, 500 mM NaCl, 250 mM imidazole, 5% Glycerol, 0.5 mM TCEP.

Procedure: The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE Healthcare). The column was washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2 : Gel filtration, Hiload 16/60 Superdex S75 prep grade, 120 ml (GE Healthcare)

GF buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP.

Procedure: The eluted fractions from the Ni-affinity Histrap column was loaded on the gel filtration column in GF buffer at 1.2 ml/min. Eluted proteins were collected in 2-ml fractions and analyzed on SDS-PAGE.

Enzymatic treatment and purification: The N-terminal His6-tag was cleaved by incubating the protein overnight with TEV protease (rotating at 4°C). Cleaved protein was purified by batch binding on 2ml pre-equilibriated 50% Ni-NTA bead solution (rotating at 4°C) for 1 hour then the column poured. Elution was done in GF buffer and eluate collected.

Extraction

Procedure
Frozen cell pellets (28g) were thawed briefly in a bath of warm water (20 ~ 37°C) then transferred to ice. One volume (i.e. 1 ml for every gram of cells) of 2x lysis buffer was added, followed by 1x lysis buffer to a total volume of 50-ml. The cells were resuspended by agitating and disrupted by high pressure homogenization (20 kpsi). Nucleic acids and cell debris were removed by adding 0.15% PEI (polyethyleneimine) from a 5% (w/v, pH 7.5) stock, stirring for 15 minutes, then centrifugation for 20 minutes at 25,000 x g . The supernatant was then further clarified by filtration (Acrodisc filters, 0.2 µm).
Concentration:The cleaved purified protein was concentrated in a VivaSpin4 (5 K MWCO) to 9.7 mg/ml and stored at 4°C. The protein concentration was determined spectrophotometrically using ε280 = 10430.
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method. The selenomethionine-labelled protein was concentrated in gel fitration buffer to a protein concentration of 9.7 mg/ml. Sitting drops comprising 135 nl of the concentrated protein mixed with 165nl of a well solution (0.8M potassium citrate, 0.1M sodium cacodylate pH 6.5) were equilibrated against well solution at 4°C. Hexagonal rods appeared within 2-3 days.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution supplemented with an additional 30% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected from a single crystal at the Se-peak (λ=0.97874Å) and at a low-energy remote (native, λ=0.98248Å) on beamline X10SA at the SLS (Swiss light source). The structure was solved using SAD and refined to 1.9Å.
Data Processing:

References

Wistow G, Bernstein SL, Wyatt MK, Behal A, Touchman JW, Bouffard G, Smith D, Peterson K (2002). Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants. Mol Vis. 8:171-84.

Bilic, I., and Ellmeier, W. (2007). The role of BTB domain-containing zinc finger proteins in T cell development and function. Immunology letters 108, 1-9.

Perez-Torrado, R., Yamada, D., and Defossez, P.A. (2006). Born to bind: the BTB protein-protein interaction domain. Bioessays 28, 1194-1202.

Xu L, Yang L, Hashimoto K, Anderson M, Kohlhagen G, Pommier Y, D'Arpa P. (2002). Characterization of BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I. BMC Genomics. 3, 1-9.