Human ankyrin repeat and SOCS box protein 9 in complex with Elongin BC

Target ID:

XX01ASB9A

Deposition Date:

Wednesday 31st March 2010

Families:

Signalling proteins

Structure type:

protein-protein

Download Coordinates:

Superceded by 3ZKJ

Authors:

J.R.C.MUNIZ,K.GUO,Y.ZHANG,V.AYINAMPUDI,P.SAVITSKY,T.KEATES,P.FILIPPAKOPOULOS,M.VOLLMAR,W.W.YUE,T.KROJER,E.UGOCHUKWU,F.VONDELFT,S.KNAPP,J.WEIGELT,C.H.ARROWSMITH,A.M.EDWARDS,C.BOUNTRA,A.BULLOCK

Site:

Oxford

2XAI

tabs

Structure Details

The ASB family belongs to the superfamily of suppressor of cytokine signaling (SOCS) box proteins and consists of 18 members in humans. Members of this family are characterised by a C-terminal SOCS box and a variable number of ankyrin repeats in the N-terminus. The SOCS box of all family members tested so far interacts with Cul5-Rbx2 as well as the elongin B-C adapter complex to form an E3 ubiquitin ligase complex. The ankyrin repeats serve as specific protein-protein interaction platforms to recruit target substrates in different biological processes (Kile et al., 2002), (Kohroki et al., 2005).

ASB9 is highly expressed in kidney and developing testis and is proposed to be a marker for spermatogenesis, although its specific role in testis has yet to be determined (Lee et al., 2008). Recently, ASB9 has been shown to interact via the ankyrin repeats with the substrate binding site of the ubiquitous mitochondrial creatine kinase, uMtCK (Kwon et al.). Both proteins co-localise in mitochondria and over-expression of ASB9 triggers ubiquitination and subsequent degradation of uMtCK resulting in negative regulation of cell growth (Kwon et al.). ASB9 is also reported to similarly target the brain-specific isoform, creatine kinase B (Debrincat et al.)

A variant of ASB9 lacking the SOCS box has been identified in human cell lines but not in human primary cells. This variant is unable to recruit the E3 ligase complex and may act as a negative regulator of ASB9. It has been speculated that this shorter form of ASB9 may play a role in tumorigenesis (Kwon et al.). Overexpression of ASB9 has been reported in prostate and breast cancer and autoantibodies against ASB9 have been detected in breast cancers but so far it is not clear if the shorter form of ASB9 may play a role in these cancers (Zhong et al., 2008).

Here we report the crystal structure of ASB9 in complex with Elongin BC at 2.60 Å resolution.

Materials and Methods

Materials and Method

Note: To our best knowledge, this should represent an accurate description of the materials and methods required to reproduce our work. If any of the content on this page is difficult to interpret or should you have trouble repeating our work, do not hesitate to contact us as soon as possible in order for us to provide additional information and advice.

Entry Clone Source: Complex

Entry Clone Accession: n/a

SGC Construct ID: XX01ASB9A-c001

GenBank GI number: n/a

Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
CTTAAGAAGGAGATATACTATGTCTGATT
GGTCTCCTATGCATGAAGCTGCAATCCAC
GGACATCAGCTGTCTCTGAGGAACCTCAT
CAGCCAGGGGTGGGCTGTGAACATCATCA
CGGCAGATCATGTTTCCCCACTCCATGAA
GCCTGTCTTGGAGGTCATCTCTCTTGTGT
GAAGATTTTATTAAAGCATGGAGCTCAGG
TGAATGGTGTGACAGCAGACTGGCACACT
CCACTGTTTAATGCTTGTGTCAGCGGCAG
CTGGGATTGTGTGAATTTGCTTCTGCAGC
ACGGAGCCAGCGTTCAACCTGAGAGTGAT
CTGGCATCCCCCATCCATGAAGCTGCTAG
GAGAGGCCACGTGGAGTGTGTCAACTCTC
TTATAGCTTATGGGGGCAACATTGACCAT
AAGATCAGCCACCTGGGCACTCCACTCTA
TTTGGCTTGTGAAAACCAACAGAGAGCCT
GTGTCAAGAAGCTTCTGGAGTCAGGAGCG
GACGTGAACCAAGGGAAAGGTCAGGATTC
CCCACTTCATGCAGTGGCCAGGACAGCCA
GTGAAGAGCTGGCCTGCCTGCTCATGGAT
TTTGGAGCGGACACCCAGGCCAAGAATGC
TGAAGGCAAACGTCCTGTGGAGCTGGTGC
CTCCAGAGAGCCCCTTGGCCCAGCTCTTC
TTGGAGAGAGAAGGGCCCCCTTCTTTGAT
GCAGTTATGCCGCCTTAGAATTCGGAAGT
GTTTTGGAATCCAGCAGCATCATAAGATA
ACCAAACTCGTCCTCCCAGAGGATCTGAA
ACAGTTTCTCCTACATCTTGCAGAGAACC
TCTACTTCCAATCGCACCATCATCACCAC
CATGATTACAAGGATGACGACGATAAGTG
AGGATCC

Final protein sequence (tag sequence in lowercase):
ASB9:
MSDWSPMHEAAIHGHQLSLRNLISQGWAVN
IITADHVSPLHEACLGGHLSCVKILLKHGA
QVNGVTADWHTPLFNACVSGSWDCVNLLLQ
HGASVQPESDLASPIHEAARRGHVECVNSL
IAYGGNIDHKISHLGTPLYLACENQQRACV
KKLLESGADVNQGKGQDSPLHAVARTASEE
LACLLMDFGADTQAKNAEGKRPVELVPPES
PLAQLFLEREGPPSLMQLCRLRIRKCFGIQ
QHHKITKLVLPEDLKQFLLHLAENLYFQ*s
hhhhhhdykddddk
ElonginC:
MMYVKLISSDGHEFIVKREHALTSGTIKAM
LSGPGQFAENETNEVNFREIPSHVLSKVCM
YFTYKVRYTNSSTEIPEFPIAPEIALELLM
AANFLDC
ElonginB:
MDVFLMIRRHKTTIFTDAKESSTVFELKRI
VEGILKRPPDEQRLYKDDQLLDDGKTLGEC
GFTSQTARPQAPATVGLAFRADDTFEALCI
EPFSSPPELPDVMKPQDSGSSANEQAVQ

Tags and additions: C-terminal his6 tag, TEV-protease cleavable (*)

Host: BL21-Elongin

Growth medium, induction protocol: A 100 ml overnight culture in LB was grown and used to inoculate 6L LB at 1:100. Plasmids were maintained by kanamycin and chloramphenicol. The cells were cultured at 37°C until the OD reached 0.78 and then decreased the temperature to 18°C. IPTG was added at 0.1mM (final concentration) and kept the culture at 18°C for overnight. The cells were harvested by centrifugation, transferred to 50 ml tubes and frozen.

Extraction buffer, extraction method: The frozen cells were thawed on ice and binding buffer added to a final volume of 150 ml. Cells were lysed using a ultrasonic processor. The lysate was centrifuged at 36,057 g for 30 minutes and the supernatant collected for purification.

Column 1: Ni-affinity, Ni-sepharose (Amersham), 8 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.

Column 1 Buffers:
Binding buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 30 mM Imidazole.
Washing Buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 30 mM Imidazole.
Elution Buffer: I: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 60 mM Imidazole.
Elution Buffer II: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 125mM Imidazole.
Elution Buffer III: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 250 mM Imidazole.

Column 1 Procedure: The column was packed by 6 ml of Ni-NTA slurry and equilibrated with 15 ml of binding buffer. The supernatant was loaded onto the column and the flow through was collected.
The column was washed with 2x20 ml of washing buffer. The protein was eluted with 10 ml of elution buffer I, II & III respectively.

Enzymatic treatment: 400 µl of TEV protease (6mg/ml) were added into the sample from Ni-NTA purification (wash II, elute I, II and. III) The sample was incubated at 4°C overnight

Column 2: Superdex 200 Hiload 16 60

Column 2 Buffers: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 0.5 mM TCEP.

Column 2 Procedure: After TEV cleavage, the sample was concentrated to 3 ml before loaded onto the AKTA Purifier. AKTA Purifier was run at 4°C. Fractions were analyzed by SDS - PAGE and the most purified fractions were collected.

Column 3: Ni-NTA

Column 3 Buffers: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 0.5 mM TCEP.

Column 3 Procedure: His-tag was cleaved by TEV protease. The sample was loaded onto the column (packed from 0.5 ml of Ni-NTA slurry). The flow through was collected and the column was then washed with 5 ml of the buffer (also collected).

Protein concentration: Samples containing the ternary complex were pooled and concentrated to 39.9 mg/ml using Centricons (3 kDa cut off). The final buffer contained 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM DTT. Protein composition was confirmed using LC-ESI MS-Tof.

Mass spectrometry characterization: Masses of purified proteins were confirmed by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% acetonitrile in water with 0.1% formic acid. For ASB9A-c009, the mass spec is 29256, the less of 130.8 as expected shows that the N-terminal methione of ASB9 is cleaved.
13132.9 for Elongin B
10963.5 for Elingin C

Crystallization & Data Collection: Crystals were obtained at 4 °C in 300 nl sitting drops set up at a ratio of 2:1 with mother liquor and 39.9 mg/ml protein. The mother liquor consisted of 0.20 M Na(malonate), 0.1M BTProp pH 6.5, 20% PEG 3350 and 10% EtGly. Resolution: 2.60 Å

References

Debrincat, M.A., Zhang, J.G., Willson, T.A., Silke, J., Connolly, L.M., Simpson, R.J., Alexander, W.S., Nicola, N.A., Kile, B.T., Hilton, D.J. (2007). Ankyrin repeat and suppressors of cytokine signaling box protein asb-9 targets creatine kinase B for degradation. J. Biol. Chem. 282, 4728-37. PubMed 17148442
Kile, B.T., Schulman, B.A., Alexander, W.S., Nicola, N.A., Martin, H.M., and Hilton, D.J. (2002). The SOCS box: a tale of destruction and degradation. Trends Biochem Sci 27, 235-241. Kohroki, J., Nishiyama, T., Nakamura, T., and Masuho, Y. (2005). ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes. FEBS Lett 579, 6796-6802. PubMed 12076535
Kwon, S., Kim, D., Rhee, J.W., Park, J.A., Kim, D.W., Kim, D.S., Lee, Y., and Kwon, H.J. (2010). ASB9 interacts ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function. BMC Biol 8, 23. PubMed 20302626
Lee, M.R., Kim, S.K., Kim, J.S., Rhim, S.Y., and Kim, K.S. (2008). Expression of murine Asb-9 during mouse spermatogenesis. Mol Cells 26, 621-624. PubMed 18776735
Zhong, L., Ge, K., Zu, J.C., Zhao, L.H., Shen, W.K., Wang, J.F., Zhang, X.G., Gao, X., Hu, W., Yen, Y., et al. (2008). Autoantibodies as potential biomarkers for breast cancer. Breast Cancer Res 10, R40. PubMed 18460216