Cryptosporidium parvum GTPase, putative Rab1a

Target ID:

cgd6_3220

Deposition Date:

Tuesday 9th October 2007

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

2RHD

Authors:

Dong, A., Wernimont, A.K., Xu, L., Lew, J., Sun, X., Khuu, C., Lin, L., Kozieradzki, I., Weigelt, J., Edwards, A.M., Arrowsmith, C.H., Bochkarev, A., Hui, R., Sukumar, D.

Site:

Toronto

ICM Datapack:

ICM Datapack

2RHD

tabs

Structure Details

Rab proteins are small-molecular-weight guanosine triphosphatases which belong to the Ras-super family.
They are involved in the regulation of vesicular transport in eukaryotic cells [1].
They act as molecular switches between a GDP-bound inactive form and GTP-bound active form in which they recruit downstream effectors onto membranes.
Rabs from different species display a significant degree of sequence conservation mainly due to homology conferred by their GTP-binding domains [2, 3].

Rab1A plays an essential role in regulating the components of the ER to Golgi vesicle transport [4].
Here we have solved the structure of Cryptosporidium parvum small GTPase Rab1A using the in situ proteolysis method [5].
The protein was mixed with the protease Chymotrypsin with a 100/1 (wt/wt) ratio prior to the crystallization setup. The proteolyzed protein crystallized in 20% PEG 3350 and 0.2M CaCl2. The mass spectrum of the crystals showed that the Chymotrypsin clipped the protein peptide between 71Phe and 72Arg.

The Crypto Rab1A structure was solved at 2.06Å.
The overall structure is similar to other known human Rab structures.
It consists of a core formed by a six stranded β-sheet surrounded by four α-helices.
The conserved phosphate-binding loop (P-loop) motif in this structure is 16GDSGVGKS23, with bound Mg (II) and GDP. Residues 68-76 of the recombinant Rab1A protein could not be located from the final electron-density map due to the proteolysis. As a result, the final structure is composed of 163 residues of the recombinant protein one GDP, three Mg2+ ions and 187 water molecules

Materials and Methods

Crystal structure of Cryptosporidium parvum small GTPase RAB1A

PDB:2RHD

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd6_3220
Entry Clone Source:
SGC Clone Accession:cgd6_3220:M1-Q174:G9
Tag:mhhhhhhssgrenlyfqg
Host:E. coli BL21-(DE3)-R3-pRARE2

Construct

Prelude:Plasmodb
Sequence:gMNPEYDYLFKLLLIGDSGVGKSCLLLRFADDTYTDSYISTIGVDFKIRTISLENKTVKLQIWDTAGQERFRTITSSYYRGAHGIIIVYDVTDRDSFDNVKQWIQEIDRYAMENVNKLLVGNKCDLVSKRVVTSDEGRELADSHGIKFIETSAKNAYNVEQAFHTMAGEIKKRVQ
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:cgd6_3220 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 2mM TCEP was added approximately 15minutes later.

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 TCEP. 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
5mMGDP5mM MgCl2 were added during concentrating the proteinMassSpec:
Crystallization:
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Zerial, M. and McBride, H. (2001). Rab proteins as membrane organizers. Nat. Rev. Mol. Cell. Biol. 2, 107-117.
Pereira-Leal, J.B. and Seabra, M.C. (2001). Evolution of the Rab family of small GTP-binding proteins. J. Mol. Biol. 234, 779-815.
Jambou, R., Zahraoui, A., Olofsson, B., Tavitian, A., Jaureguiberry, G. (1996). Small GTP-binding proteins in Plasmodium Falciparum. Biol. Cell. 88, 113-121.
Ward,G.E. Tilney, L.G., Langsley, G., (1997). RabGTPases and the unusual secretory pathways of Plasmodium. Parasitol. Today 13, 57-62.
Aiping Dong, Xiaohui Xu, Aled M Edwards, In situ proteolysis for protein crystallization and structure determination. Nature Methods, online 4 November 2007, pp 1-3.