Toxoplasma gondii ubiquitin-conjugating enzyme E2-25, E2 domain

Target ID:

TgTwinScan_2721

Deposition Date:

Thursday 24th November 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2F4Z

Authors:

A.DONG,J.LEW,Y.ZHAO,M.MELONE,Z.ALAM,I.KOZIERADSKI,G.WASNEY,M.VEDADI,M.SUNDSTROM,J.WEIGELT,A.M.EDWARDS,C.H.ARROWSMITH,A.BOCHKAREV,R.HUI,T.HILLS,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2F4Z

tabs

Structure Details

Toxoplasma gondii is an apicomplexan parasite and the causative agent for toxoplasmosis,
with reportedly over 50 million people infected per year in North America alone via cat feces, raw meat or blood transfusion.
An infected person with a healthy immune system typically does not show any obvious symptoms.
On the other hand, those who are immuno-compromised, including for example HIV patients or recipients of transplanted organs, suffer swollen lymph glands, muscle pains and possibly damage to eyes and even the brain in acute cases.

The T. gondii genome encodes a number of ubiquitin conjugating enzymes,
which are involved in protein ubiquitylation,1,2,3
a universal pathway used to mark proteins for degradation and other forms of regulation.
One such E2 enzyme, TgTwinScan_2712, is 45% identical to the
human E2-25 protein, also known as HIP2.
Therefore, we dub this protein Tg-E2-25, which, like E2-25 from other organisms,
consists of a UBA domain as well as the catalytic E2 domain.

Our structure of Tg-E2-25 includes only the catalytic domain
and features the highly conserved E2 fold of a beta strand core flanked by N- and C-terminal alpha helices.

Tg-E2-25: Toxoplasma gondii ubiquitin conjugating enzyme E2-25 (TgTwinScan_2721)

PDB:2F4Z

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:TgTwinScan_2721
Entry Clone Source:Toxoplasma gondii cDNA from the lab of Prof. David Sibley
SGC Clone Accession:TgTwinScan_2721:M1-A172; plate MAC00T:G10
Tag:N-terminal: N-terminal His-tag with integrated TEV protease site (*): MGSSHHHHHHSSGRENLYFQ*G
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqgMATAQPRGTPREQARLLKELADIQQLQRAHDSEPAATHSTSHGVSAQIVGGDIHRWRGFIAGPLGTPYEGGHFTLDIVIPPDYPYNPPKMKFVTKIWHPNISSQTGAICLDILKHEWSPALTIRTALLSIQAMLADPVPTDPQDAEVAKMMIENHPLFVQTAKLWTETFAKE
Vector:p15-Tev-LIC

Growth

Medium:Terrific Broth (TB)
Antibiotics:50 microG/mL kanamycin and 25 microG/mL chloramphenicol
Procedure:A single colony was inoculated into 10 mL of LB with of Antibiotics and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of TB with Antibiotics in a 250 mL shaking flask and incubated at 37 degC for 3 hours. The culture was then transferred into 1.8 L of above-specified growth medium with Antibiotics and 0.3 mL of antifoam (Sigma) in a 2L bottle and cultured using the LEX system to an OD600 of 2.5, cooled to 15 degC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

Purification

Procedure
The cleared cell lysate was loaded onto a DE52 (Whatman) column packed with 10 g of resin (previously activated with 3 M NaCl and equilibrated with Binding Buffer), and subsequently onto a 2.5 mL Ni-NTA column at approximately 1.5 mL/min. When all the lysate was loaded, 20 mL of Binding Buffer was added to the DE52 column. Then the Ni-NTA column was washed with 200 mL of Wash Buffer at 2Â2.5 mL/min. After washing, the protein was eluted from the Ni-NTA column with 15 mL of Elution Buffer. EDTA was added immediately to 1 mM. DTT was then added to 1 mM 15 minutes later.

The protein was dialysed overnight in a dialysis cassette (Pierce) in Crystal Buffer. The following day, it was concentrated using a 15 mL Amicon Ultra centrifugal filter device Millipore (5 kD cutoff).Protein concentration was estimated by means of absorbance at OD280. Aliquots of the purified protein were flash frozen in N2(l) and stored at -80°C.

Extraction

Procedure
Cells were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with protease inhibitor (1 mM benzamidine-HCl and 1 mM phenylmethyl sulfonyl fluoride, PMSF). Resuspended pellets stored at -80 degC were thawed overnight at 4 degC on the day before purification. Prior to mechanical lysis, each pellet from 1 L of culture was pretreated with 0.5% CHAPS and 500 units of benzonase for 40 minutes at room temperature. Cells were mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18,000 psi. The cell lysate was centrifuged at ~75,000 x g for 20 minutes at 10 degC.
Concentration:
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of sitting drop vapor diffusion in Intelli-Plate. The plate was set with 0.5 microL protein and 0.5 microL buffer in each drop, and 100 microL reservoir volume per well. Crystals grew in 31% PEG 1500, 0.2 M MgCl2, 0.1 M tris and pH 8.4 with spermidine as an addiive at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Passmore, L. and Barford, D. (2004) Getting into position: the catalytic mechanisms of protein ubiquitylation Biochem. J. (2004) 379 (513-525)

2. Wu, P. Y., Hanlon, M., Eddins, M., Tsui, C., Rogers, R. S., Jensen, J. P., Matunis, M. J., Weissman, A. M., Wolberger, C. P. and Pickart, C. M. (2003) A conserved catalytic residue in the ubiquitin-conjugating enzyme family. EMBO J. 22, 5241-5250

3. Picart, C. M. (2001) Mechanisms underlying ubiquitination. Annu. Rev. Biochem (70):503-33.