Cryptosporidium parvum 14-3-3 protein epsilon in complex with phosphorylated peptide

Target ID:

cgd3_1290

Deposition Date:

Friday 27th October 2006

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2NPM

Authors:

A.DONG,J.LEW,G.WASNEY,H.REN,L.LIN,A.HASSANALI,W.QIU,Y.ZHAO,D.DOYLE,M.VEDADI,I.KOEIERADZKI,A.M.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,A.BOCHKAREV,R.HUI,S.BROKX,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2NPM

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 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

cgd3_1290, which is 64% identical to the nearest human isoform, 14-3-3 ε.
Similar to most others, this Cp-14-3-3 structure features a homodimer, with each monomer comprising the characteristic nine helices.
Unlike the first Cp-14-3-3 structure solved by the SGC, this dimer conforms to the vice-grip geometry of
all pre-existing 14-3-3 structures.
A phosphopeptide, RAI(pS)LP, added before crystallization, is also bound at the peptide binding site of this Cp-14-3-3 ε structure.

Cp 14-3-3 ε

PDB:2NPM

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd3_1290 (CryptoDB.org)
Entry Clone Source:Cp gDNA Iowa strain
SGC Clone Accession:
Tag:N-Terminal hexahistidine tag with integrated tobacco etch virus (TEV) protease cleavage site.
Host:E. coli BL21 (DE3) Rosetta-R3

Construct

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

Growth

Medium:
Antibiotics:
Procedure:Cryptosporidium parvum putative 14-3-3 protein was expressed in E. coli BL21 (DE3) Oxford in Terrific Broth (TB) in the presence of ampicillin and chloramphenicol (100 µg/mL and 34 µg/mL respectively). A single colony was inoculated into 50 mL of LB with ampicillin and chloramphenicol in a 125 mL flask and incubated with shaking at 250 rpm overnight at 37 ºC. The culture was transferred into 2 X 1.8 L TB with ampicillin and chloramphenicol and 0.3 mL of antifoam (Sigma) in 2 L bottles and cultured using the LEX system to an OD600 of 4.0. The culture was cooled to 15 ºC, and isopropyl-1-thio-D-galactopyranoside (IPTG) was added to 0.4 mM, and the culture was incubated overnight at 15 ºC.

Purification

Procedure
Column 1: The cleared cell lysate was loaded onto a column containing 10 g DE-52 resin (Whatman), and then directly onto a 2.5 mL Ni-NTA (Qiagen) column at approximately 1.5 mL/min. When all the lysate was loaded, the two column system was washed with 15 mL binding buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM imidazole, and 5 % glycerol) at 2 mL/min. After washing, the protein was eluted from the Ni-NTA column with 15 mL of Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, and 5 % glycerol). EDTA was added immediately to 1 mM; and DTT was added to 1 mM 15 minutes later.

TEV Protease Cleavage: The eluted Cp 14-3-3 protein was treated with TEV protease in a 1:500 molar ratio of protease:protein, and incubated at 4°C overnight concurrent with dialysis against 10 mM HEPES, pH 7.5, 500 mM NaCl, and 5 mM imidazole. The cut protein was separated from the uncut protein by passage through another 2.5 mL Ni-NTA column.

Column 2: The cut Cp 14-3-3 was applied to a Sephadex S200 26/60 gel filtration column (GE Healthcare) pre-equilibrated with 10 mM HEPES, pH 7.5 and 500 mM NaCl. The collected fractions corresponding to the eluted protein peak were concentrated to 10 mg/mL using a 15 mL Amicon Ultra centrifugal filter device (Millipore). Aliquots of the purified Cp 14-3-3 protein were stored at -70 °C.

Extraction

Procedure
The culture was harvested by centrifugation and the cell pellet was suspended in 160 mL of binding buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, and 5 mM imidazole) with protease inhibitor (1 mM benzamidine-HCl and 1 mM phenylmethyl sulfonyl fluoride, PMSF) and kept in 50 mL Falcon tubes at Â 80 ºC. Before purification, the cell suspension was thawed overnight at 4 ºC. Prior to mechanical lysis, each tube of cell suspension was pretreated with 0.5 % CHAPS and 500 units of benzonase (per 40 mL of resuspended cell pellet) for 40 minutes at room temperature. Then the cells were mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18000 psi. The lysate was centrifuged at 24000 rpm for 20 minutes at 10 ºC.
Concentration:10 mg/mL
Ligand
MassSpec:
Crystallization:Purified Cp 14-3-3 was first treated with 2 mM DTT and a three fold molar excess of consensus peptide 2 Â RAI(pS)LP where pS denotes a phosphoserine Â followed by incubation on ice for 1 h. The protein:peptide complex was crystallized using the hanging drop vapour diffusion method in a 24-well Linbro plate. The protein solution (1 µL) was added to 1 µL reservoir buffer containing 14% PEG3350, 0.1 M Calcium Acetate, 0.2 M trimethylamine-N-oxide, and 0.1 M HEPES pH 7.0, placed over 500 µL reservoir buffer, and incubated for at 293 K. Diamond shaped crystals (100-200 µm) grew to maximum size in two 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.