Human protein 14-3-3 gamma isoform

Target ID:

YWHAGA

Aliases:

14-3-3GAMMA

Deposition Date:

Tuesday 13th September 2005

Families:

Signalling proteins

Related Diseases:

CancerNeurobiologySignalling

Structure type:

apo

Download Coordinates:

2B05

Authors:

E.PAPAGRIGORIOU,J.ELKINS,C.ARROWSMITH,Y.ZHAO,E.J.DEBRECZENI,A.EDWARDS,J.WEIGELT,D.DOYLE,F.VON DELFT,A.TURNBULL,X.YANG

Site:

Oxford

ICM Datapack:

ICM Datapack

2B05

tabs

Structure Details

Protein phosphorylation by kinases is a major process of instigating and transmitting
cellular signals.
One potential consequence of the addition of the phosphate group to the target protein
is that it forms a handle for other protein such as the 14-3-3 protein to grab. We have determined the structure of the gamma isoform of 14-3-3 bound to a phosphorylated peptide taken from the apoptosis sign-regulating kinase 1.

Creutzfeldt-Jakob disease is diagnosed on the basis of clinical findings and the presence
of 14-3-3gamma in the patient's CSF (Peoc'h et al 2005).
Specifically, the presence of 14-3-3 gamma in the CSF is a marker for sporadic CJD
(Van Everbroeck et al, 2005).
When arterials are damaged 14-3-3 gamma was shown to be up-regulated (Autieri, 2004).
In human lung cancer. the levels of 14-3-3 gamma plus 3 other isoforms are greatly increased
as compared to the equivalent normal tissues (Qi et al, 2005).
In one cell line (HEK293) under standard conditions, a total of 170 binding partners
were identified that clustered into proteins involved in regulation of the cytoskeleton,
membrane signalling and cell fate (Jin et al, 2004).
These results suggest that 14-3-3gamma plays a significant role in normal cell function
as well as the coordinated response of a cell to damage.

Materials and Methods

YMHAG

PDB:2B05

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:YWHAGA-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:C-terminal hexahistidine tag
Host:BL-21(DE3)R3 phage resistant

Construct

Prelude:
Sequence:MVDREQLVQKARLAEQAERYDDMAAAMKN VTELNEPLSNEERNLLSVAYKNVVGARRS SWRVISSIEQKTSADGNEKKIEMVRAYRE KIEKELEAVCQDVLSLLDNYLIKNCSETQ YESKVFYLKMKGDYYRYLAEVATGEKRAT VVESSEKAYSEAHEISKEHMQPTHPIRLG LALNYSVFYYEIQNAPEQACHLAKTAFDD AIAELDTLNEDSYKDSTLIMQLLRDNLTL WTSDQQDDDGGEGNNENLYFQ
Vector:pTvHR21- SGC

Growth

Medium:
Antibiotics:
Procedure:Freshly transformed E. coli cells was used to inoculate 1 litre of TB plus 100 mg/ml ampicillin. When OD600 reached ~0.5 the temperature was shifted down from 37°C to 25°C for 1 hour before induction with the addition of 1 mM IPTG. Protein expression was allowed to carry on for a futher 4 hours before harvest.

Purification

Procedure
Column 1 : Ni-affinity, HisTrap, 1 ml (GE/Amersham)
The cell extract was loaded on the column at 0.8 ml/min on an AKTA-express system (GE/Amersham). The column was then washed with 10 column volumes of WB1 and then eluted with EB at 0.8 ml/min. The eluted peak of A280 was automatically collected. Column 2 : Gel filtration, Hiload 16/60, S75 16/60 - 120 ml
The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions. Using a Centricon 10 K cutoff concentrator the YWHAGA pooled fractions was concentrated to 25 mg/ml. Concentration was determined from the absorbance at 280 nm.

Enzymatic treatment : Gel filtration fractions containing YWHAGA were pooled , concentrated to 5 mg/ml using a Vivaspin 10kD cutoff spin concentrator. Next TEV protease cleavage was applied (30µl of in-house purified TEV per ml). TEV digestion was carried out at 4 ° C overnight. The following steps were carried out to remove the cleaved products and TEV protease: Place 200 µl of 50 % Ni-NTA agarose in a 1.5 ml eppendorf tubes, add 1ml of 50 mM Tris pH 8.0, 150 mM NaCl mix, spin down and remove buffer. Repeat this resin wash step once. Add the TEV treated protein sample to the resin and mix for 30 min. Finally spin down resin and collect the supernatant which contains the cleaved YWHAGA.

Extraction

Procedure

Concentration:
Ligand
MassSpec:Before His-Tag removal
Expected MWt.: 30249.3
Measured MWt.: 30249 (main); 30118

After Hit-Tag removal
Expected MWt.: 29097.1
Measured MWt.: 29097.0
Crystallization:The purified TEV cleaved YWHAQA was then concentrated to 40 mg/ml and distributed into 30 x 50 µl aliquots and frozen at -80 ° C. First crystallisation attempts indicated that the protein concentration was too high therefore the sample was diluted to 28 mg/ml using GF buffer. YWHAGA was crystallised in the presence of a phosphorylated peptide. The peptide RSIpSLP where pS stands for a phosphorylated Serine was synthesised by Thermo Electron Corporation (www.thermo.com). The peptide was dissolved to a concentration of 40 mM in water. Before crystallisation set-up YWHAGA was mixed with the peptide to a molar ratio of 3:1 peptide-to-YWHAGA. Crystals grew from a 1:1 ratio mix of YWHAGA/peptide-to-reservoir ( 25 % PEG3350, 0.1 Bis/Tris pH 5.5, 0.2 M MgCl2).
NMR Spectroscopy:
Data Collection:Resolution: 2.55 Å; X-ray source: Synchrotron SLS -X10, single wavelength.
Data Processing:

References

Autieri MV. (2004). Inducible expression of the signal transduction protein 14-3-3 gamma in injured arteries and stimulated human vascular smooth muscle cells. Exp. Mol. Pathol. 76: 99-107.

Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD, Pawson T. (2004). Proteomic, functional, and domain-based analysis of
in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Curr. Biol. 14: 1436-1450.

Peoc'h K, Schroder HC, Laplanche J, Ramljak S, Muller WE. (2005). Determination of 14-3-3 protein levels in cerebrospinal fluid from Creutzfeldt-Jakob patients by a highly sensitive capture assay. Neurosci. Lett. 301: 167-170.

Qi W, Liu X, Qiao D, Martinez JD. (2005). Isoform-specific expression of 14-3-3 proteins in human lung cancer tissues. Int. J. Cancer. 113: 359-363.

Van Everbroeck BR, Boons J, Cras P. (2005). 14-3-3 {gamma}-isoform detection distinguishes sporadic Creutzfeldt-Jakob disease from other dementias. J. Neurol. Neurosurg. Psychiatry. 76: 100-102.