Plasmodium falciparum ubiquitin-conjugating enzyme variant

Target ID:

PFC0255c

Deposition Date:

Tuesday 22nd May 2007

Families:

MalariaUbiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2Q0V

Authors:

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

Site:

Toronto

2Q0V

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, members of the these three enzyme classes and related homologues are also involved in other regulatory events in the cell.

Two such homologues are the closely related UBC variants Mms2 and Uev1a, which lack the active site cysteine required for UBC activity. In humans, Uev1a and Mms2 can each form a stable heterodimeric complex with Ubc13. 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. In this study, we have determined the structure of PFC0255c, the only Plasmodium falciparum homologue of Mms2/Uev1a, by x-ray crystallography. It adopts a structure very similar to human Uev1a determined at the SGC (PDB ID 2A4D), with a four stranded antiparallel beta sheet alongside three alpha helices. This structure will help to lead to a better understanding of ubiquitylation mediated events in P. falciparum, the major causative agent of human malaria worldwide.

Pf-UEV1: Plasmodium falicparum homologue of ubiquitin conjugating enzyme E2 variant UEV1

PDB:2Q0V

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFC0255c
Entry Clone Source:
SGC Clone Accession:PFC0255c:I5-N142; plate MAC02D::D12
Tag:N-terminal His6-tag with integrated TEV cleavage site (*): mhhhhhhssgrenlyfq*g
Host:E. coli BL21-(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:gIVPRSFRLLDELERGQKGNVSEGVSFGLESADDITLSNWSCTIFGQPGTVFENRIYSLTIFCDDNYPDSPPTVKFDTKIEMSCVDNCGRVIKNNLHILKNWNRNYTIETILISLRQEMLSSANKRLPQPNEGEVYSNN
Vector:pET15-MHL

Growth

Medium:TB
Antibiotics:100 microG/mL ampicillin and 34 microG/mL chloramphenicol
Procedure: 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 ~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 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 1-1.5 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 2-2.5 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 12 mg/mL using a 15 mL Amicon Ultra centrifugal filter device (Millipore) with a cutoff matching the MW of the protein. The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE. The concentrated protein was 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 sonicated for 6 minutes and the cell lysate was centrifuged using a Beckman JLA-16.250 rotor at ~38,400 x g (16,000 rpms) for 45 minutes at 4 degC.
Concentration:
Ligand
MassSpec:
Crystallization:The protein was crystallized at 20 degC in 1.4 M Na/KPO4, pH 7.0, 1 mM DTT, with 1 microL buffer added to 1 microL protein 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.
Eddins, M.J., Carlile, C.M., Gomez, K.M., Pickart, C.M., and Wolberger, C. (2006) Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat. Struct. Mol. Biol. 13 (10): 915-920.
Philip, N., and Haystead, T. A. (2007) Characterization of a UBC13 kinase in Plasmodium falciparum. PNAS 104 (19): 7845-7850.