Cryptosporidium parvum Vacuolar Protein Sorting 29

Target ID:

Cp-PF14_0064

Deposition Date:

Tuesday 21st June 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2A22

Authors:

S.BROKX,Y.ZHAO,Z.ALAM,J.LEW,J.WEIGELT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,R.HUI,J.R.WALKER,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2A22

tabs

Structure Details

Cryptosporidium parvum is a protozoan parasite from the phylum Apicomplexa,
which also includes Plasmodium species and Toxoplasma gondii.
It is the causative agent for cryptosporidiosis in humans and animals,
and may be transmitted via contaminated water and food, as well as contact with infected animals and humans.
Collectively, apicomplexans represent a major health concern worldwide.

Typical symptoms are cramps, fever, diarrhea and weight loss,
but may be more serious and particularly persistent in patients with compromised immune systems,
such as those with AIDS or cancer.

We have determined the structure of the vacuolar protein sorting protein 29 (Vsp29)
from Cryptosporidium parvum,
namely cgd7_2060.
This protein is an ortholog of PF14_0064 from the malaria parasite Plasmodium falciparum, with the two proteins sharing 49% amino acid sequence identity. This protein is also expressed in Plasmodium berghei and Plasmodium yoelii - both rodent parasites.Vsp29 is involved in the trafficking of proteins to different compartments in the eukaryotic cell,
a set of critical and complex processes which have been studied in yeast and human cells.
Vsp29 is one protein subunit of a large multimeric complex termed the retromer complex.1
The retromer complex is involved in the retrograde transport of other cargo proteins, themselves chaperone proteins,
from prevacuolar endosomes back to the trans Golgi network.

The crystal structure of human Vsp29, 46% identical to Cp-Vsp29, has recently been determined.2
This structure was used as a starting model in order to determine the structure of Cp-Vsp29.
The two proteins have very similar structures,
comprised of two β-sheets (one five- and one six-stranded), and three β-helices,
together adopting a four-layered α-β-β-β sandwich fold.

Materials and Methods

Cp-Vsp29: Cryptosporidium parvum vacuolar protein sorting 29

PDB:2A22

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd7_2060
Entry Clone Source:Cryptosporidium parvum strain Iowa genomic DNA
SGC Clone Accession:CP-PF14_0064; plate MS:A10
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPR*GS
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsSSTDFGDLVLLIGDLKIPYGAKELPSNFRELLATDKINYVLCTGNVCSQEYVEMLKNITKNVYIVSGDLDSAIFNPDPESNGVFPEYVVVQIGEFKIGLMHGNQVLPWDDPGSLEQWQRRLDCDILVTGHTHKLRVFEKNGKLFLNPGTATGAFSALTPDAPPSFMLMALQGNKVVLYVYDLRDGKTNVAMSEFSK
Vector:pET28a-LIC

Growth

Medium:Terrific Broth (TB)
Antibiotics:
Procedure:A 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 2.5, cooled to 15 degC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 degC.

Purification

Procedure
The cleared cell lysate was loaded onto a column containing 10 g DE-52 resin (Whatman) anion exchangeresin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer), and then directly onto a 3 mL Ni-NTA (Qiagen) column at approximately 1.5 mL/min. When all the lysate was loaded, 20 mL of Binding Buffer was added to the DE52 column. Then the Ni-NTA column was washed with 200 mL of Wash Buffer.After washing, the protein was eluted from the Ni-NTA column with 15 mL of Elution Buffer. EDTA was added immediately to 1 mM.DTT was added to 5 mM 15 minutes later.

The eluted protein was applied to a Sephadex S200 26/60 gel filtration column (GE Healthcare) pre-equilibrated with Gel Filtration Buffer.The collected fractions corresponding to the eluted protein peak were concentrated to 7 mg/mL using a 15 mL Amicon Ultra centrifugal filter device (5 kD cutoff). The concentrated sample was flash frozen in N₂(l) and 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 mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18,000 psi. The cell lysate was centrifuged at ~75,000 x g for 20 minutes at 10 degC.
Concentration:27 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of hanging drop vapor diffusion in a Linbro plate. The plate was set with 1 microL protein (27 mg/mL) and 0.75 microL buffer in each drop, and 500 microL reservoir volume per well. Crystals grew in 22% (w/v) polyethylene glycol (PEG) 4000, 100 mM sodium citrate and 200 mM ammonium acetate at pH 5.6 and 18 degC in 5 days.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. C. R. Haft, M. de la Luz Sierra, R. Bafford, et al (2000) Mol. Cell Biol. 11 (12) 4105-16.

2. D. Wang, M. Guo, Z. Liang, et al (2005) J. Biol. Chem. 280 (24) 22962-7.