Toxoplasma gondii specific mitochondrial acyl carrier protein

Target ID:

59.m03510

Deposition Date:

Thursday 28th February 2008

Families:

Lipid signalling

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

3CE7

Authors:

Wernimont AK, Dong A, Cheng Y, Khuu C, Lin YH, Lam A, Brand V, Kozieradzki I, Cossar D, Arrowsmith CH, Bountra C, Weigelt J, Edwards AM, Bochkarev A, Hui R, Qiu W

Site:

Toronto

3CE7

tabs

Structure Details

An essential metabolic pathway in all organisms is fatty acid synthesis (FAS). Since fatty acids are poorly soluble in water, they are produced with the fatty acyl chain attached to an acyl carrier protein (ACP). There are two types of FAS pathways found in nature, which differ fundamentally in structure and mechanism. Type I FAS is found in most eukaryotes, including humans, while type II FAS is found in plants and prokaryotes (bacteria). Interestingly, apicomplexan parasites adopt diverse strategies. Plasmodium falciparum, the major causative agent of human malaria, employs an apicoplast-based type II FAS. Type I FAS takes place in the cytoplasm of Cryptosporidium parvum, the parasite responsible for cryptosporidiosis in humans and farm animals.

Adding to the intrigue is the apparent existence in Toxoplasma gondii of an apicoplast type II pathway and a mitochondrial type I pathway. The structure of the mitochondrial isoform of Tg-mACP is presented here.

Materials and Methods

Tg-mACP

PDB:3CE7

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:59.m03510
Entry Clone Source:
SGC Clone Accession:59.m03510:A136-Q241:B2-MAY6XA:B1-2
Tag:mgsshhhhhhssgrenlyfqg
Host:BL21-(DE3)-Ros-Ox

Construct

Prelude:
Sequence:ANETTKQESPVVDTDINAVTNYIVGMCQKFLQKGEKVTPSSKLEELRTREDRLWDCLDTVEFVLDVEEIFDVTVPDEVADNFQTLQEIADFVVSERAKAGKFMKDQ
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:Tg-mACP was expressed in E. coli BL21-(DE3)-Rosetta Oxford resistant 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 degC. The culture was transferred into 1.8 L of TB with ampicillin/chloramphenicol (100 microgram/mL and 34 microgram/mL respectively) and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD600 of 4.5, 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 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 âÂÂ80 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.
Concentration:
Ligand
MassSpec:
Crystallization:MAY6XA:B1-21.5 M NH4SO4, 0.2 M K,Na Tartrate, 0.1 M Na Acetate pH 4.5 + 15% glycerol
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Mazumdar J, Wilson EH, Masek K, Hunter CA, and Striepen B (2006). Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. PNAS vol. 103, no. 35.
Surolia A, Ramya TNC, Ramya V, and Surlia N (2004). 'FAS't inhibition of malaria. Biochem. J. 383.
Roujeinikova A, Baldock C, Simon WJ, et al. (2002). X-Ray crystollagraphic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site. Structure 10.