Human Supressor of Cytokine Signalling-2 (SOCS2) in complex with Elongin C and Elongin B

Target ID:

XX10SOCS2A

Deposition Date:

Wednesday 14th December 2005

Families:

SOCS-box-containing

Related Diseases:

NeurobiologySignallingCancerMetabolism

Structure type:

protein-protein complex

Download Coordinates:

2C9W

Authors:

J.E.DEBRECZENI,A.BULLOCK,A.AMOS,P.SAVITSKY,A.BARR,N.BURGESS,M.SUNDSTROM,J.WEIGELT,C.ARROWSMITH,A.EDWARDS,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2C9W

tabs

Structure Details

The suppressor of cytokine signalling (SOCS) family comprises SOCS1-7 and CIS1 (1). Members of this family have been shown to be negative regulators of cytokine receptor signalling via the JAK/STAT pathway (2). The 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 (3).

The role of SOCS2 in growth hormone (GH) signalling in vivo has been convincingly
demonstrated by the phenotype of SOCS2 deficient mice,
which are 30-40% larger than normal littermates (4).
This phenotype is similar to mice overexpressing GH and human patients with gigantism.
The specific linkage of the SOCS2 -/- phenotype to the JAK/STAT pathway has been demonstrated
by STAT5b/SOCS2 double knockout mice which have no overgrowth phenotype (5).
Further studies have demonstrated specific binding of SOCS2 to the phosphorylated GH receptor
and regulation of its signalling in vitro (6).

The structure of human SOCS2 in complex with ElonginC and ElonginB was refined at 1.9 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

SOCS2-ElonginC-ElonginB complex

PDB:2C9W

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|4507263
Entry Clone Source:MGC
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m); N-terminal his6 tag, TEV-protease cleavable (*)
Host:BL21(DE3)

Construct

Prelude:
Sequence:SOCS2:
mhhhhhhssgvdlgtenlyfqsmQAARLAKALRELGQTGWYWGSMTVNEAKEKLKEAPEGTFLIRDSSHSDYLLTISVKTSAGPTNLRIEYQDGKFRLDSIICVKSKLKQFDSVVH LIDYYVQMCKDKRTGPEAPRNGTVHLYLTKPLYTSAPSLQHLCRLTINKCTGAIWGLPLPTRLKDYLEEYKFQV
ElonginC:
MMYVKLISSDGHEFIVKREHALTSGTIKAMLSGPGQFAENETNEVNFREIPSHVLSKVCMYFTYKVRYTNSSTEIPEFPIAPEIALELLMAANFLDC
ElonginB:
MDVFLMIRRHKTTIFTDAKESSTVFELKRIVEGILKRPPDEQRLYKDDQLLDDGKTLGECGFTSQTARPQAPATVGLAFRADDTFEALCIEPFSSPPELPDVMKPQDSGSSANEQAVQ
Vector:pLIC-SGC (SOCS2), pACYCDUET-1 (ElonginBC)

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure
Column 1: Ion exchange - Nucleic acid removal - DEAE cellulose (DE52, Whatmann), 25 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, then washed with 20 ml binding buffer prior to loading the sample.
Buffers: Binding buffer
Procedure: Supernatant was applied at gravity flow, followed by a wash with 50 ml binding buffer. The column flow-through was collected.

Column 2: Ni-affinity - Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.
Buffers : Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol; Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5% glycerol; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol
Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 8 x 10 ml wash buffer at 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.
Enzymatic treatment : (Histag cleavage) Samples containing the SOCS2-ElonginC/B complex were pooled and 20 µg TEV protease added for overnight incubation at 4°C.
Column 3: HiLoad S75 Gel Filtration
Procedure: Protein was ran in 25 mM HEPES pH 7.5, 250 mM NaCl and the ternary complex fractions pooled.

Column 4: Ion exchange Mono Q column .
Buffers: A: 50 mM Hepes pH 7.5; B: 50 mM Hepes pH 7.5, 2M NaCl
Procedure: SOCS2-ElonginC/B complex was applied to a 1 ml MonoQ column in buffer A and eluted from the column by a linear gradient with buffer B.
Concentration : Samples containing the ternary complex were pooled and concentrated in Centricons (10 kDa cut off) to 22 mg/ml. Protein composition was confirmed us ing LC-ESI MS-Tof.

Extraction

Buffers
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 225 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:
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°C in 1 µl sitting drops using a precipitant of 0.08 M Na-Cacodylate pH 6.5, 0.16 M NaCl, 1.6 M (NH4)2SO4.
NMR Spectroscopy:
Data Collection:Data were collected at the SLS. The structure was solved by molecular replacement using 1LM8.
Data Processing:

References

Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, (1997). Metcalf D, Nicola NA, Hilton DJ. 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.
Metcalf D, Greenhalgh CJ, Viney E, Willson TA, Starr R, Nicola NA, Hilton DJ, Alexander WS. (2000). Gigantism in mice lacking suppressor of cytokine signalling-2. Nature 405 (6790):1069-73.
Greenhalgh CJ, Bertolino P, Asa SL, Metcalf D, Corbin JE, Adams TE, Davey HW, Nicola NA, Hilton DJ, Alexander WS. (2002). Growth enhancement in suppressor of cytokine signaling 2 (SOCS-2)-deficient mice is dependent on signal transducer and activator of transcription 5b (STAT5b). Mol Endocrinol. 16(6):1394-1406.
Greenhalgh CJ, Rico-Bautista E, Lorentzon M, Thaus AL, Morgan PO, Willson TA, Zervoudakis P, Metcalf D, Street I, Nicola NA, Nash AD, Fabri LJ, Norstedt G, Ohlsson C, Flores-Morales A, Alexander WS, Hilton DJ. (2005). SOCS2 negatively regulates growth hormone action in vitro and in vivo. J Clin Invest. 115(2):397-406.