Plasmodium falciparum ubiquitin-conjugating enzyme, putative

Target ID:

PFE1350c

Deposition Date:

Monday 20th August 2007

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2R0J

Authors:

Wernimont, A.K., Lew, J., Lin, L., Hassanali, A., Kozieradzki, I., Zhao, Y., Schapira, M., Bochkarev, A., Weigelt, J., Sundstrom, M., Arrowsmith, C.H., Edwards, A.M., Hui, R., Brokx, S.

Site:

Toronto

ICM Datapack:

ICM Datapack

2R0J

tabs

Structure Details

Ubiquitylation of a target protein, a pathway common to all eukaryotes, requires the participation of ubiquitin, a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (UBC; E2), and a ubiquitin-protein ligase (E3). After attachment of uibiquitin residue(s), the target protein is destined for degradation by the proteasome. Apart from this canonical role of the ubiquitylation pathway, the members and other homologues are also involved in other regulatory events in the cell.

One such homologue is UBC13, which, in humans and yeast, forms a heterodimeric complex with an inactive UBC, either Uev1a or Mms2. These heterodimeric complexes help mediate Lys63-linked polyubiquitin chain formation on target proteins, and through these modifications a number of cellular events, such as DNA damage repair and NF-κB transcription factor activation, can be regulated (1). In Plasmodium falciparum, the protist parasite which is the major causative agent of human malaria, a putative UBC13 homologue has been identified (PlasmoDB ID PFE1350c). A recent report has described a novel atypical protein kinase in P falciparum, PfPK9, which phosphorylates and thereby inhibits the activity of PFE1350c (2). In this study, we have determined the structure of PFE1350c by x-ray crystallography. It adopts a structure very similar to other UBCs, with a four stranded antiparallel beta sheet alongside three alpha helices. It is hoped that this structure will help contribute to a better understanding of ubiquitin mediated events in P. falciparum, and perhaps lead to the development of new antimalarial drugs.

Putative ubiquitin conjugating enzyme, PFE1350c, from Plasmodium falciparum

PDB:2R0J

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFE1350c
Entry Clone Source:
SGC Clone Accession:PFE1350c:I3-N150:D3
Tag:mhhhhhhssgrenlyfqg
Host:Ros-Ox

Construct

Prelude:
Sequence:IPRRITKETQNLANEPPPGIMAVPVPENYRHFNILINGPDGTPYEGGTYKLELFLPEQYPMEPPKVRFLTKIYHPNIDKLGRICLDILKDKWSPALQIRTVLLSIQALLSSPEPDDPLDSKVAEHFKQDKNDAEHVARQWNKIYANNN
Vector:p15-tev-lic

Growth

Medium:TB
Antibiotics:
Procedure:PFE1350c was expressed in E. coli BL21-(DE3)-Rosetta-Oxford cells in Terrific Broth meida in the presence of carbenicillin/chloramphenicol (100 microg/mL and 34 microg/mL respectively). A single colony was inoculated into 5 mL of LB with of carbenicillin/chloramphenicol (100 microg/mL and 34 microg/mL respectively) in a 14 mL round bottom tube and incubated with shaking at 250 rpm overnight at 37 degC. The culture (0.5 mL) was transferred into 50 mL of TB with carbenicillin and chloraphenicol in a 250 mL shaking flask and incubated at 37 degC overnight. The culture was then transferred into 2 X 1.8 L of TB with ampicillin and chloramphenicol and 0.3 mL of antifoam (Sigma) in 2 L bottles and cultured using the LEX system to an OD600 of ~5, cooled to 15 degC, and protein expression was induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

Purification

Procedure
The cleared lysate was loaded onto a column prepacked with 10 g DE52 (Whatman) anion exchange resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer); and subsequently onto a 2 mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer at approximately 1 Â 1.5 mL/min. After the lysate was loaded, the DE52 was further washed with 10 mL of Binding Buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2 Â 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer. EDTA was immediately added to the elution fraction to 1 mM; and DTT was added to 2 mM after 15 minutes.

The sample was loaded onto a Sephadex S200 26/60 column equilibrated with Gel Filtration Buffer. The fractions from the peak corresponding to monomeric protein were pooled and concentrated to 10 mg/mL using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE. The concentrated protein was stored at -80 degC.

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 4 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with the addition of protease inhibitors (1 mM benzamidine 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 18000 psi; and the cell lysate was centrifuged using a Beckman JA-25.50 rotor at ~75000 x g (24000 rpm) for 20 minutes at 10 degC.
Concentration:
Ligand
MassSpec:
Crystallization:MAY5GB:D4-23.5 M Sodium Formate, 0.1 M Bis-Tris, pH 6.0The protein was crystallized at 20 degC in 3.5 M Sodium Formate, 0.1 M Bis-Tris, pH 6.0 using the hanging drop vapour diffusion method.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Andersen, P.L., Zhou, H., Pastushok, L., et al (2005) Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J. Cell Biol. 170 (5): 745-755.
Philip, N., and Haystead, T. A. (2007) Characterization of a UBC13 kinase in Plasmodium falciparum. PNAS 104 (19): 7845-7850.