Toxoplasma gondii Ubiquitin Conjugating Enzyme E2

Target ID:

TgTwinScan_2218

Deposition Date:

Thursday 8th September 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2AYV

Authors:

W.TEMPEL,A.DONG,Y.ZHAO,J.LEW,Z.ALAM,M.MELONE,G.WASNEY,I.KOZIERADZKI,M.VEDADI,C.ARROWSMITH,M.SUNDSTROM,J.WEIGELT,A.EDWARDS,A.BOCHKAREV,R.HUI,M.AMANI,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2AYV

tabs

Structure Details

Toxoplasma gondii is an apicomplexan organism 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.

We have solved the structure of a putative ubiquitin-conjugating enzyme from T. gondii,
namely
TgTwinScan_2218.
It is a homolog of human UBE2E2,
with the two proteins sharing 60% sequence identity and hard to distinguish structures,
as well as of
UBC4 from yeast.
TgTwinScan_2218 is also the ortholog of Plasmodium falciparum ubiquitin-conjugating enzyme
PFL0190w,
with 82% sequence identity.

In eukaryotic cells, protein degradation and many other cell cycle regulation processes
are achieved by covalently attaching one or more ubiquitins to the substrate
(e.g. for recognition by the 26S proteasome in the case of proteolysis).
This ubiquitylation process is catalyzed successively by a ubiquitin activating enzyme (E1),
a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligases (E3).1,2,3
Specifically, E1 binds ubiquitin via a thioester bond and transfers it onto E2,
which in turn passes it onto the substrate protein in coordination with E3.

The E2 enzyme class is the second most abundant of the three
and structurally the most studied group
in the ubiquitylation pathway.1,2,3
This Tg-UBC structure does not deviate from the highly conserved fold
consisting of a core of beta strands flanked by N- and C-terminal alpha helices.
Furthermore, the structure also harbors another defining E2 feature:
The catalytic cysteine (CYS85) which forms a thioester bond with ubiquitin is located in
a groove on the loop between the beta strand mesh and the ensuing helix.

Materials and Methods

Tg-UBC: Toxoplasma gondii ubiquitin conjugating enzyme (TgTwinScan_2218)

PDB:2AYV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:TgTwinScan_2218
Entry Clone Source:Toxoplasma gondii cDNA from the lab of Prof. David Sibley
SGC Clone Accession:TgTwinScan_2218:A2-A146; plate MAC00T:D2
Tag:N-terminal His-tag with integrated TEV protease site: MGSSHHHHHHSSGRENLYFQ*G
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqgALKRINKELNDLSKDPPTNCSAGPVGDDMFHWQATIMGPEDSPYSGGVFFLNIHFPSDYPFKPPKVNFTTKIYHPNINSQGAICLDILKDQWSPALTISKVLLSISSLLTDPNPDDPLVPEIAHLYKSDRMRYDQTAREWSQKYA
Vector:p15TV-L

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 2.5 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 2 Â 2.5 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 in a dialysis cassette (Pierce) overnight dialysis against Crystal Buffer. The following day it was concentrated using a 15 mL Amicon Ultra centrifugal filter device from Millipore (10 kD cutoff). Protein concentration was measured by means of absorbance at OD280. The concentrated protein was flash frozen in N2(l) in 150 microL aliquots and stored at -80 degC.

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:26 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of sitting drop vapor diffusion in a 96-well Intelli-Plate. The plate was set with 1 microL cleaved protein and 1 microL buffer in each drop, and 100 microL reservoir volume per well. Crystals grew in two days in 3.5M Na formate, 0.1M NaOAc pH 4.6 at 20 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.