Plasmodium falciparum ubiquitin conjugating enzyme Pf-UBC13 in complex with Pf-Uev1a

Target ID:

PP-UBC13

Deposition Date:

Thursday 21st August 2008

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Parasitic disease

Structure type:

protein-protein

Download Coordinates:

3E95

Authors:

Wernimont AK, Lam A, Ali A, Brokx S, Lin L, Zhao Y, Lew J, Ravichandran M, Wasney G, Vedadi M, Kozieradzki I, Schapira M, Bochkarev A, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Hui R, Qiu W, Brand VB

Site:

Toronto

3E95

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.

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 this Pf-UBC13 enzyme (2). Furthermore, there is also a homologue of Uev1a/MMs2, namely the protein encoded by the gene PFC0255c. We have previously solved the structures of Pf-UBC13 and Pf-Uev1a. Here we present the complex of the two.

Materials and Methods

Pf-UBC13

PDB:3E95
SGC Clone Accession:PFE1350c:I3-L152:B3 & PFC0255c:M1-S140:C12
Tag:N-terminal tag: mgsshhhhhhssgrenlyfqg
Host:BL21-(DE3)-V2R-pRARE2
Vector:pET15-MHL

Sequence:
PFE1350c: IPRRITKETQNLANEPPPGIMAVPVPENYRHFNILINGPDGTPYEGGTYKLELFLPEQYPMEPPKVRFLTKIYHPNIDKLGRICLDILKDKWSPALQIRTVLLSIQALLSSPEPDDPLDSKVAEHFKQDKNDAEHVARQWNKIYANNNVL
PFC0255c: MSEVIVPRSFRLLDELERGQKGNVSEGVSFGLESADDITLSNWSCTIFGQPGTVFENRIYSLTIFCDDNYPDSPPTVKFDTKIEMSCVDNCGRVIKNNLHILKNWNRNYTIETILISLRQEMLSSANKRLPQPNEGEVYS

Growth

Medium:TB
Procedure:Both PfUBC13 and PfUev1a were expressed in E. coli BL21-(DE3)-V2R-pRARE2 strain in Terrific Broth (TB) in the presence of ampicillin/chloramphenicol (100 microgram/mL and 34 microgram/mL respectively). A single colony was inoculated into 100mL of LB with of ampicillin/chloramphenicol (100 microgram/mL and 34 microgram/mL respectively) in a 250 mL baffled flask and incubated with shaking at 250 rpm overnight at 37 °C. The culture was transferred into 4.0 L of TB with ampicillin/chloramphenicol (100 microgram/mL and 34 microgram/mL respectively) and 0.15 mL of antifoam (Sigma) in a 1 L bottle and cultured using the LEX system to an OD600 of 5-6, cooled to 15 °C, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 °C.

Purification

Procedure: Both PfUBC13 and PfUev1a were purified separately using the same protocol as described below: The cleared lysate was loaded onto a 1.0-2.5 mL Ni-NTA (Qiagen) column (pre-equilibrated with Binding Buffer) at approximately 1.5-2.0 mL/min. The Ni-NTA column was then washed with 150 mL of Wash Buffer at 2-2.5 mL/min. After washing, the protein was eluted with Elution Buffer. The eluted sample was applied to a Sephadex S200 26/60 gel filtration column pre-equilibrated with Gelfiltration Buffer. The fractions corresponding to the eluted protein peak were collected and further concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE gel. The concentrated protein was stored at 4 degC. For long term storage, the protein was flash frozen and 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 sonication, each pellet from 1 L of culture was pretreated with 0.5 % CHAPS and 500 units of benzonase for 40 minutes at room temperature. After 10 minutes sonication, the cell lysate was centrifuged using a Beckman JLA-16.250 rotor at 15,500 rpms for 45 minutes at 4 degC.

Structure Determination

Crystallization:0.5 M Mg Formate 20 % ethylene glycol; Two proteins were mixed in 1:1 molar ration prior to crystallization. Hanging drop. 293K.

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.