Plasmodium falciparum nucleolar GTP-binding protein 1

Target ID:

PFF0625w

Deposition Date:

Friday 3rd August 2007

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

2QU8

Authors:

Wernimont A, Lew J, Lin L, Arrowsmith CH, Edwards AM, Weigelt J, Sundstrom M, Bochkarev A, Hui R, Qiu W, Sukumar D, Hassanali A

Site:

Toronto

ICM Datapack:

ICM Datapack

2QU8

tabs

Structure Details

The G-protein superfamily clan includes the following Pfam members: septin, Ras, NOG1, MMR_HSR1, miro, IIGP, GTP_EFTU, G-alpha, Dynamin_N, SRPRB, DUF258, ATP_bind_1, Arf and AIG1.
The NOG1 family includes eukaryotic, bacterial and archaeal proteins.
Its domain represents a conserved region of approximately 60 residues in length.
In Saccharomyces cerevisiae, the NOG1 gene has been shown to be essential for cell viability, suggesting that NOG1 may play an important role in nucleolar functions1. In particular, NOG1 is believed to be functionally linked to ribosome biogenesis 2, which occurs in the nucleolus 3.
In eukaryotes, NOG1 mutants have been found to disrupt the biogenesis of the 60S ribosomal subunit.
The DRG and OBG proteins as well as the prokaryotic NOG-like proteins are homologous throughout their length to the amino half of eukaryotic NOG1, which contains the GTP binding motifs; the N-terminal GTP-binding motif is required for function.
We have solved the first structure of NOG1 from Plasmodium Falciparum (PFF0625w) in complex with GDP and MgCl2.
NOG1 is structurally similar (22% identity) to a conserved protein TT1381 from Thermus thermophilus HB8 (PDB: 1UDX). Like other GTPase family, the overall structure shows a characteristic nucleotide binding fold consisting of a seven-stranded β-sheet surrounded by 5 α-helices with a tightly bound GDP at the active site.
The C-terminal end has distinctive 8-turn α-helices which may play crucial roles for the potential specific binding partner recognition.

Materials and Methods

Crystal Structure of Nucleolar GTP-binding protein 1 from Plasmodium Falciparum

PDB:2QU8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFF0625w
Entry Clone Source:
SGC Clone Accession:PFF0625w:L164-N370; plate MAC00G:G3
Tag:mgsshhhhhhssgrenlyfq
Host:Ros-Ox

Construct

Prelude:
Sequence:gLPSINPHKKTIILSGAPNVGKSSFMNIVSRANVDVQSYSFTTKNLYVGHFDHKLNKYQIIDTPGLLDRAFENRNTIEMTTITALAHINGVILFIIDISEQCGLTIKEQINLFYSIKSVFSNKSIVIGFNKIDKCNMDSLSIDNKLLIKQILDNVKNPIKFSSFSTLTGVGVEQAKITACELLKNDQAESILLDQEQLLNTKLGETKN
Vector:p15-tv-lic

Growth

Medium:TB
Antibiotics:
Procedure:PFF0625w was expressed in E. coli BL21-(DE3)-Rosetta-Oxford cells in Terrific Broth (TB) in the presence of ampicillin/chloramphenicol (50 microg/mL and 25 microg/mL respectively). A single colony was inoculated into 10 mL of LB with of ampicillin/chloramphenicol (50 microg/mL and 25 microg/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of TB with 50 microg/mL ampicillin in a 250 mL shaking flask and incubated at 37 degC for 3 hours. Then the culture was transfered into 1.8 L of TB with 50 microg/mL Chloramphenicol and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD 600 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 Ni-NTA (Qiagen) column (pre-equilibrated with Binding Buffer) at approximately 2Â 3 mL/min. The volume of the Ni-NTA resin was pre-determined by the predicted protein yield from test expression analysis. After the lysate was loaded, the Ni-NTA column was 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 eluted fraction to 1 mM; and DTT was added to 1mM after approximately 15 more minutes.

The eluted sample from Ni-NTA was loaded onto a Sephadex S75 26/60 column pre-equilibrated with Gel Filtration Buffer (10mM HEPES, pH7.5, 500mM NaCl).The fractions corresponding to the eluted protein peak were collected. The His-tag was cleaved with TEV protease for 3hr at room temperature in the presence of DTT. Mass spectrometry results confirmed fully cleaved His-tag protein. The cleaved sample was applied on 1mL Ni-NTA column pre-equilibrated with Binding Buffer (no glycerol). The flow-through was collected and the column was rinsed with additional 5mL of binding buffer.The collected sample was concentrated using a 15 mL Amicon Ultra centrifugal filter device. The protein sample identity and purity were evalulated by mass spectroscopy and SDS-PAGE gel.The concentrated protein was stored at -80deg C.

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 lysed by sonicating for 10min and was centrifuged using a Beckman JA-16.25 rotor at 15,500 rpms for 45 minutes at 4 degC.
Concentration:
Ligand
1mM GDP2mM MgCl2 were added during concentrating the proteinMassSpec:
Crystallization:MAY52M_S:H1015%glycerolThe protein was crystallized at 20 degC in 28%PEG2K MME, 0.1MBis-Tris pH6.5 using the sitting drop method.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Park JH, Jensen BC, Kifer CT, Parsons M (2001) J. Cell Sci. 114:173-185.
Bryan CJ, Qin W, Charles TK, Parsons M (2003) J. of Biol. Chem. 34: 32204-32211.
Kallstrom G, Hedges J, Johnson A (2003) Molec. and cellular Biol. 23: 4344-4355.