Plasmodium yoelii ubiquitin-conjugating enzyme, putative

Target ID:

PY01609

Deposition Date:

Friday 11th May 2007

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2PWQ

Authors:

W.QIU,A.DONG,A.HASSANALI,L.LIN,S.BROKX,S.ALTAMENTOVA,T.HILLS,J.LEW,M.RAVICHANDRAN,I.KOZIERADZKI,Y.ZHAO,M.SCHAPIRA,A.M.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,A.BOCHKAREV,R.HUI,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

2PWQ

tabs

Structure Details

Plasmodium yoelii is a malaria pathogen that infects rodents and often serves as a model organism for the study of human malaria pathogens.
The Plasmodium genomes encode 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, encoded by the gene PY01609, is 47% identical to the human E2-25 protein, also known as HIP2. Therefore, we dub this protein Py-E2-25, which, like E2-25 from other organisms, consists of a UBA domain as well as the catalytic E2 domain.

Our structure of Py-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.
We have also solved
the structure of the
Toxoplasma gondii ortholog of this protein.

Materials and Methods

Py-E2-25: Plasmodium yoelii ubiquitin conjugating enzyme E2-25 (PY01609)

PDB:2PWQ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PY01906
Entry Clone Source:Plasmodium yoelii genomic DNA
SGC Clone Accession:PY01609:s:S8-S202:D11; plate MAC01G:D11
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:mgsshhhhhhssgrenlyfqgSKELLRLQKELKDIENENVQEIDAHIKDSNFFEWVGFIKGPEGTPYEGGHFTLAITIPNDYPYNPPKIKFVTKIWHPNISSQTGAICLDVLKNEWSPALTIRTALLSIQALLSDPQPDDPQDAEVAKMYKENHALFVKTASVWTKTFATGPKEEPREVIIKKITEMGFSEDQAKNALIKANWNETLALNTLLENS
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 20% PEG 3350, 0.2 M sodium dihydrogen phosphate at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Passmore, L. and Barford, D. (2004) Getting into position: the catalytic mechanisms of protein ubiquitylation Biochem. J. (2004) 379 (513-525)
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
Picart, C. M. (2001) Mechanisms underlying ubiquitination. Annu. Rev. Biochem (70):503-33.