Human SPRY domain-containing SOCS box protein SSB-2 in complex with peptide

Target ID:

SPSB2A

Aliases:

GRCC9MGC2519SPSB2SSB-2SSB2

Deposition Date:

Thursday 25th September 2008

Families:

SOCS-box-containingUbiquitin Related BiologyUbiquitylation - E3 Ligases

Structure type:

protein-ligand

Download Coordinates:

3EMW

Authors:

Filippakopoulos, P., Sharpe, T., Keates, T., Murray, J.W., Savitsky, P., Roos, A., Pike, A.C.W., Von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S., Bullock, A.

Site:

Oxford

ICM Datapack:

ICM Datapack

3EMW

tabs

Structure Details

Human SPRY-domain-containing SOCS box protein 2 (SPSB2 or SSB-2) belongs to a family of four proteins containing an N-terminal SPRY (or B30.2) domain and a C-terminal SOCS box (SPSB1-4). The SOCS box functions as an adaptor domain that binds ElonginBC and mediates further assembly with cullin ubiquitin ligases (Kile et al., 2002). The SPRY domain was first identified in the dual-specificity kinase splA and ryanodine receptors and is proposed to function as a protein-protein recognition domain. Several human diseases arise from mutations in genes coding for SPRY domain containing proteins, for example mutations in the MEFV gene are associated with familial Mediterranean fever (Masters et al., 2006).

Knockout mice of SPSB2 have a reduced rate of platelet production, resulting in very mild thrombocytopenia and show low levels of blood urea nitrogen (BUN) pointing to a unique role of SPSB2 in regulating platelet production and BUN levels (Masters et al., 2005). The SPRY domain mediates interaction with the MET receptor tyrosine kinase and prostate apoptosis response protein 4 (Wang et al., 2005) (Masters et al., 2006). Here we determined the crystal structure of the SPSB2 SPRY domain in complex with a peptide derived from the RNA helicase VASA. Binding is dependent on four SPSB2 loops which hydrogen bond to an Asp-Ile-Asn-Asn-Asn motif in VASA.

Materials and Methods

Entry clone source: MGC

Entry clone accession: IMAGE:3546861

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Final protein sequence:
mhhhhhhssgvdlgtenlyfq*sMPEGLE
ELLSAPPPDLGAQRRHGWNPKDCSENIEV
KEGGLYFERRPVAQSTDGARGKRGYSRGL
HAWEISWPLEQRGTHAVVGVATALAPLQT
DHYAALLGSNSESWGWDIGRGKLYHQSKG
PGAPQYPAGTQGEQLEVPERLLVVLDMEE
GTLGYAIGGTYLGPAFRGLKGRTLYPAVS
AVWGQCQVRIRYLGE

Host: BL21(DE3)-R3-pRARE2

Growth medium, induction protocol: Starter cultures in LB media (50 ml LB, 50 µg/ml kanamycin/34 µg/ml chloramphenicol) were inoculated from a glycerol stock and grown overnight. Three flasks containing 1L LB/antiobiotic media were each inoculated with 10 ml of overnight culture and grown at 37°C, 160 rpm untilOD₆₀₀ = 0.5. The temperature was then reduced to18oC at which point protein expression was induced with 0.5 mM IPTG (final concentration). Cells were harvested the following morning by centrifugation (15 min, 5000rpm, 4°C). The pellets were each resuspended in 40 ml binding buffer and frozen at ‑20°C. Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol.

Extraction method: Frozen pellets were thawed and fresh 1 mM PMSF and 0.5 mM TCEP were added. Cells were lysed using by sonication. DNA was precipitated by addition of PEI (polyethyleneimine) to a final concentration of 0.15%. The lysate was then centrifuged at 16,500 rpm for 60 minutes and the supernatant collected for purification.

Protein Purification, Column 1: IMAC Sepharose 6 Fast Flow resin:
Column 1: Ni-affinity. Ni-sepharose (Amersham), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.

Procedure: The soluble lysate was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 100 ml wash buffer under gravity flow. The protein was eluted by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM and 250 mM); fractions were collected until essentially all protein was eluted. 5 mM DTT was added for overnight storage together with TEV protease for cleavage of the N-terminal hexahistidine tag (however, mass spec and SDS PAGE analysis showed later that the tag was resistant to TEV cleavage and remained intact).

Buffers : Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol; Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% glycerol; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol.

Column 2: Gel Filtration, HiLoad 16/60 Superdex 200 prep grade column run on an AKTA-prime system.

Buffers: Gel Filtration Buffer: 25mM Hepes pH 7.5, 250 mM NaCl.

Procedure (Gel Filtration): The protein solution was concentrated to 3ml using a 5 kD MWCO Amicon Ultra concentrator. Protein was injected onto a pre-equilibrated gel filtration column and run at a flow rate of 1.0 ml/min. Fractions containing SPSB2 were pooled and mixed with 10 mM DTT for overnight storage.

Concentration: The buffer was adjusted to 20 mM HEPES pH 7.4, 150 mM NaCl, 10 mM DTT. Protein was concentrated to 5 mg/ml determined by UV absorbance at 280 nm using an extinction coefficient of 47900 M^-1cm^-1. Then a 5 mM solution of VASA peptide (DINNNNNIVEDVERKREFYI) was added and the protein-peptide mix concentrated further to 9 mg/ml prior to crystallization. A little precipitate was observed from insoluble peptide and was removed by centrifugation.

Mass spec characterization: LC- ESI -MS TOF. Expected mass: 23819 Da. The expected mass was observed.

Crystallization: Crystals were obtained using the vapour diffusion method by mixing 666 nl of the concentrated protein (9 mg/ml) with 333 nl of a well solution containing the following components: 0.22M Na/KPO4, 11% ethylene glycol, 22% PEG 3350. Crystallization experiments were setup at 4°C.

Data Collection: Crystals were cryo-protected using the well solution supplemented with an additional 25% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected from a single crystal at the Diamond synchrotron (UK) beamline I04. The structure was solved by molecular replacement and refined to 1.8 Å.

References

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 in biochemical sciences 27, 235-241.

Masters, S.L., Palmer, K.R., Stevenson, W.S., Metcalf, D., Viney, E.M., Sprigg, N.S., Alexander, W.S., Nicola, N.A., and Nicholson, S.E. (2005). Genetic deletion of murine SPRY domain-containing SOCS box protein 2 (SSB-2) results in very mild thrombocytopenia. Molecular and cellular biology 25, 5639-5647.

Masters, S.L., Yao, S., Willson, T.A., Zhang, J.G., Palmer, K.R., Smith, B.J., Babon, J.J., Nicola, N.A., Norton, R.S., and Nicholson, S.E. (2006). The SPRY domain of SSB-2 adopts a novel fold that presents conserved Par-4-binding residues. Nature structural & molecular biology 13, 77-84.

Wang, D., Li, Z., Messing, E.M., and Wu, G. (2005). The SPRY domain-containing SOCS box protein 1 (SSB-1) interacts with MET and enhances the hepatocyte growth factor-induced Erk-Elk-1-serum response element pathway. The Journal of biological chemistry 280, 16393-16401.