Human SOCS4-ElonginC-ElonginB complex

Target ID:

XX02SOCS4B

Deposition Date:

Wednesday 26th July 2006

Families:

SOCS-box-containingUbiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

Signalling

Structure type:

protein-protein complex

Download Coordinates:

2IZV

Authors:

J.E.DEBRECZENI,A.BULLOCK,E.PAPAGRIGORIOU,A.TURNBULL,A.C.W.PIKE,F.GORREC,F.VON DELFT,M.SUNDSTROM,C.ARROWSMITH,J.WEIGELT,A.EDWARDS,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2IZV

tabs

Structure Details

The suppressor of cytokine signalling (SOCS) family comprises in human SOCS1-7 and CIS1. In general SOCS proteins contain a variable N-terminal domain, a central SH2 domain and a C-terminal SOCS box. The SOCS box shows weak sequence similarity with the alpha domain of the von Hippel Lindau (VHL) tumour suppressor which binds ElonginC/B to target hypoxia-inducible factor (HIF-1 a) for proteasomal degradation. By analogy it has been speculated that many, if not all, SOCS box-containing proteins act as part of an E3 ubiquitin ligase complex.

The role of SOCS4 in vivo is not clear and so far no substrates have been unambiguously identified. SOCS4 shares significant sequence homology with SOCS5 and the lack of an obvious lymphoid phenotype in SOCS5-deficient mice may reflect functional redundancy between these two SOCS family members. Both SOCS proteins are induced by EGF and are reported to target EGFR for proteasomal degradation. The structure of human SOCS4 in complex with ElonginC and ElonginB was refined at 2.55 Å resolution and includes a central SH2 domain flanked by an N-terminal extended SH2 subdomain and a C-terminal SOCS box.

Materials and Methods

SOCS4-ElonginC-ElonginB complex

PDB:2IZV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|40807480
Entry Clone Source:
SGC Clone Accession:
Tag:N-terminal his6 tag, TEV-protease cleavable (*) however tag cannot be cleaved due to steric hindrance
Host:BL-21(DE3)R3 phage resistant

Construct

Prelude:
Sequence:SOCS4: mhhhhhhssgvdlgtenlyfqs*MLVPDLLQINNNPCYWGVMDKYAAEALLEGKPEGTFLLRDSAQEDYLFSVSFRRYSRSLHARIEQWNHNFSFDAHDPCVFHSPDITGLLEHYKDPSACMFFEPLLSTPLIRTFPFSLQHICRTVICNCTTYDGIDALPIPSSMKLYLKEYHYKSKVRVLRIDAPE
ElonginC:
MMYVKLISSDGHEFIVKREHALTSGTIKAMLSGPGQFAENETNEVNFREIPSHVLSKVCMYFTYKVRYTNSSTEIPEFPIAPEIALELLMAANFLDC
ElonginB:
MDVFLMIRRHKTTIFTDAKESSTVFELKRIVEGILKRPPDEQRLYKDDQLLDDGKTLGECGFTSQTARPQAPATVGLAFRADDTFEALCIEPFSSPPELPDVMKPQDSGSSANEQAVQ
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:A 50 ml overnight culture in LB was grown and used to inoculate 6L TB at 1:300. Plasmids were maintained by kanamycin and chloramphenicol. Cultures were grown at 37 degC until they reached an OD 600 of 0.5 and then cooled to 20 degC. Expression was induced overnight for ~14 hours using 0.5 mM IPTG at an induction OD 600 of 1.0. The cells were harvested by centrifugation, transferred to 50 ml tubes and frozen.

Purification

Procedure
Column 1: Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatmann), 50 g of resin divided in two 2.5 x 20 cm columns. The resin was hydrated in 2.5M NaCl, then washed with 20 ml binding buffer prior to loading the sample.
Procedure: The column was equilibrated with binding buffer. The lysate was applied to the column which was subsequently washed with wash buffer 1. CSNK1G3 was eluted with elution buffer. The eluted protein was collected and analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 5mM. The N-terminal his 6 -tag was cleaved by incubating the protein overnight with TEV protease, shrimp alkaline phosphatase and lambda phosphatase.
Column 2: Ni-affinity
Ni-sepharose (Amersham), 8 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.
Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 200 ml wash buffer under gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted. After elution DTT was added to a final concentration of 10 mM.
Column 3: HiLoad 16/60 S200 Gel Filtration
Procedure: Protein was applied in 25 mM HEPES pH 7.5, 250 mM NaCl and the ternary complex fractions pooled.
Column 4: Ion exchange Source 15Q column
Procedure: SOCS4-ElonginC/B complex was applied to a 10 ml Source15Q column in buffer A and eluted from the column by a linear gradient with buffer B . Elution occurred at ~250 mM NaCl.

Extraction

Procedure
The frozen cells were thawed on ice and binding buffer (plus 1 mM PMSF, 0.5 mM TCEP) added to a final volume of 350 ml. Cells were lysed using a high pressure cell disruptor. The lysate was centrifuged at 17,000 RPM for 30 minutes and the supernatant collected for purification.
Concentration:Samples containing the ternary complex were pooled and concentrated to 11 mg/ml using Centricons (10 kDa cut off). The final buffer contained 50 mM HEPES pH 7.5, 250 mM NaCl, 10 mM DTT. Protein composition was confirmed using LC-ESI MS-Tof.
Ligand
MassSpec: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.
Crystallization:Crystals were obtained at 4 degC in 150 nl sitting drops set up at a ratio of 1:1 with mother liquor and 11 mg/ml protein. The mother liquor consisted of 2M NaCl, 10% PEG6000.
NMR Spectroscopy:
Data Collection:
Data Processing:Data were collected at the SLS beamline 10. The structure was solved by molecular replacement using the SOCS2 complex determined previously at the SGC (pdbcode 2C9W).

References

Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ. (1997). A family of cytokine-inducible inhibitors of signalling. Nature 387(6636):917-921.
Kubo M, Hanada T, Yoshimura A. Suppressors of cytokine signaling and immunity. (2003). Nat Immunol. 12:1169-1176.
Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, Hilton DJ. (2002). The SOCS box: a tale of destruction and degradation. Trends Biochem Sci. 27(5):235-241.
Brender C, Columbus R, Metcalf D, Handman E, Starr R, Huntington N, Tarlinton D, Ã˜dum N, Nicholson SE, Nicola NA, Hilton DJ, Alexander WS (2004). SOCS5 Is Expressed in Primary B and T Lymphoid Cells but Is Dispensable for Lymphocyte Production and Function. Mol Cell Biol. 24(13): 6094-6103.