Cryptosporidium parvum 14-3-3 protein

Target ID:

cgd7_2470

Deposition Date:

Tuesday 12th December 2006

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2O8P

Authors:

A.DONG,J.LEW,G.WASNEY,L.LIN,A.HASSANALI,Y.ZHAO,M.VEDADI,I.KOZIERADZKI,A.M.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,J.R.WALKER,A.BOCHKAREV,R.HUI,S.J.BROKX,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2O8P

tabs

Structure Details

14-3-3 proteins are important regulatory proteins found in all eukaryotes. They participate in protein-protein interactions with other phosphorylated proteins. Through these interactions, 14-3-3 proteins help regulate a wide variety of cellular processes such as cell division, metabolic reactions, and cell signaling. There are seven closely related 14-3-3 paralogues found in humans (β, γ, ε, ζ, η, τ and σ) which have partially overlapping functions. The structures of all seven of these proteins have been determined,
including many by the SGC.

Cryptosporodiosis is a primarily water-borne disease common in the third world, and is caused by the protist parasite Cryptosporidium parvum.
The C. parvum genome contains at least three genes encoding 14-3-3 proteins, which have poor sequence conservation with each other and relative to their homologues in other organisms.
To wit, only one of these three proteins is more than 30% identical in sequence to any of the human isoforms.
Little is known about these C. parvum 14-3-3 proteins as there is scant literature on protozoan 14-3-3 proteins in general.
We have determined the structure of the C. parvum 14-3-3 protein
cgd7_2470,
which is 27% identical overall to the nearest human isoform, 14-3-3 ε,
and 18.1% identical overall to the nearest C. parvum isoform,
cgd3_1290. Despite this low sequence homology, the cgd7_2470 protein adopts a similar structure to other 14-3-3 proteins, with nine α-helices arranged to form a shallow pocket for binding other proteins in vivo.

Cp 14-3-3: Cryptosporidium parvum 14-3-3 protein

PDB:2O8P

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd7_2470
Entry Clone Source:
SGC Clone Accession:cgd7_2470:E6-K238; MAC01E:E4
Tag:
Host:E. coli BL21-(DE3)-R3-pRARE2

Construct

Prelude:
Sequence: gEMDERLLQKYRAQVFEWGGCFDKMFEALKSLIYLSEFENSEFDDEERHLLTLCIKHKISDYRTMTSQVLQEQTKQLNNDELVKICSEYVFSLRKDIKAFLQSFEDCVDRLVEKSFFSKFFKLKVKSDISRYKLEFGLCSLEDSKKIHQDAFTLLCEHPDKIEQLPLGFIQNLAYILSEKYGEKKQVFNMLNSLGKILELQIKEQENMDRKAQITVYLQGIKDYIEK
Vector:p15-TEV-LIC

Growth

Medium:M9 SeMet media kit from Medicilon
Antibiotics:
Procedure:A single colony was inoculated into 10 mL of LB with of ampicillin/chloramphenicol (100 microG/mL and 34 microG/mL respectively) and incubated with shaking at 250 rpm overnight at 37 degC. The culture was transferred into 50 mL of TB with ampicillin/chloramphenicol (100 microG/mL and 34 microG/mL respectively) 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 ampicillin/chloramphenicol (100 microG/mL and 34 microG/mL respectively) and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD600 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 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 2.5 mL Ni-NTA (Qiagen) column at approximately 1.5 mL/min. When all the lysate was loaded, both columns were washed with 15 mL binding buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2 mL/min. 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 2 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 fractions corresponding to the eluted protein peak were collected.

The eluted protein was treated with TEV protease in a molar ratio of protease:protein determined based on measured activity of the available TEV,with the addition of 1 mM tris(2-carboxyethyl) phosphine (TCEP) and 15 mM imidazole, overnight at 4 degC. The cleaved protein was separated from the uncleaved protein by passage through another 2.5 mL Ni-NTA column.

TEV Protease Cleavage: The eluted Cp 14-3-3 protein was treated with TEV protease in a 1:200 molar ratio of protease:protein with the addition of and 15 mM imidazole, overnight at 4degC. The cut protein was separated from the uncut protein by passage through another 2.5 mL Ni-NTA column.The buffer of the cleaved protein was exchanged against the Crystal Buffer and the protein was concentrated to 8 mg/mL using a 15 mL Amicon Ultra centrifugal filter device from Millipore (5 kD cutoff). Aliquots of the purified protein were stored at -80 degC.

Extraction

Procedure
Pellets from 4 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Bufferwith 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 18000 psi; and the cell lysate was centrifuged using at ~75000 x g for 20 minutes at 10 degC.
Concentration:8 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means by hanging drop vapor diffusion in a 24-well Linbro plate. The plate was set with 1 microL cleaved native protein (8 mg/mL) pre-treated with 2 mM DTT and 1 microL buffer in each drop, and 500 microL reservoir volume per well. Cubic crystals grew to maximum size (100 microM) in 15% PEG3350, 0.3 M ammonium acetate and 0.2 M sodium citrate at pH 5.6 at 4 degC after 3 days.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA, Deng M, Liu C, Widmer G, Tzipori S, Buck GA, Xu P, Bankier AT, Dear PH, Konfortov BA, Spriggs HF, Iyer L, Anantharaman V, Aravind L, Kapur V. (2004) Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science. 304(5669):441-5.
Aitken A (2006). 14-3-3 proteins: a historic overview. Semin Cancer Biol. 16(3):162-72.
Al-Khedery B, Barnwell JW, Galinski MR (1999). Stage-specific expression of 14-3-3 in asexual blood-stage Plasmodium. Mol Biochem Parasitol. 102(1):117-30.
Inoue M, Nakamura Y, Yasuda K, Yasaka N, Hara T, Schnaufer A, Stuart K, Fukuma T (2005). The 14-3-3 proteins of Trypanosoma brucei function in motility, cytokinesis, and cell cycle. J Biol Chem. 280(14):14085-96.
Assossou O, Besson F, Rouault JP, Persat F, Ferrandiz J, Mayencon M, Peyron F, Picot S (2004). Characterization of an excreted/secreted antigen form of 14-3-3 protein in Toxoplasma gondii tachyzoites. FEMS Microbiol Lett. 234(1):19-25.