Cryptosporidium parvum 14-3-3 protein

Target ID:

cgd1_2980

Deposition Date:

Saturday 30th September 2006

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

Superceded by 3EFZ

Authors:

A.K.WERNIMONT,A.DONG,W.QIU,J.LEW,G.A.WASNEY,M.VEDADI,I.KOZIERADZKI,Y.ZHAO,H.REN,Z.ALAM,L.Y.LIN,M.SUNDSTROM,J.WEIGELT,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,R.HUI,S.BROKX,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2IJP

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 putative C. parvum 14-3-3 protein cgd1_2980, which is 27% identical to the nearest human isoform, 14-3-3 ζ. Similar to most others, this Cp-14-3-3 structure features a homodimer, with each monomer comprising the characteristic nine helices. Whereas the monomers in the structure of the human β isoform feature a rigid rotation with respect to each other in the N-terminus, the two monomers dimer in our Cp-14-3-3 structure also differ in the same region, with one monomer more disordered than the other.

Cp 14-3-3

PDB:2IJP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd1_2980 (CryptoDB.org)
Entry Clone Source:
SGC Clone Accession:
Tag:N-terminal His6-tag with integrated TEC cleavage site (*): mgsshhhhhhssglvpr*gs
Host:E. coli BL21-(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqgITEKNMKLSEGAYRAKLADMVGNYKDVIKVLTESSDFRDNSLILLLAGSLRNRVTSIRNSLKSIKSQEEKLRKEKSLNNEFIQVIEDIKRDFEESILLESEDVIRIIDDNLLMYSEEGARAFCIKLKGDLMRYKAEILKDEEKNQCIKQAVEFYEDALQRERSFLEKYPSDPLYLATILNYTILKYDLLGNPEGAMKFANRAIQAAENSRSDSEQFSENTEKLLKILRDNVSQWEQGCSGLLTSAFF
Vector:p15TV-L

Growth

Medium:TB
Antibiotics:100 microG/mL ampicillin and 34 microG/mL chloramphenicol
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 ~5, cooled to 15 degC and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 ºC.

Purification

Procedure
The cleared lysate was loaded onto a column prepacked with 10 g DE52 (Whatman) anion exchange resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer); and subsequently onto a 1.0 Â 2.5 mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer at approximately 1 Â 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; and DTT was added to 1 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 20 mg/mL using a 15 mL Amicon Ultra centrifugal filter device (Millipore). Aliquots of the purified protein were stored at -80 °C.

Extraction

Procedure

Concentration:20 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized using the hanging drop vapour diffusion method in a 24-well Linbro plate. For the purpose of removing partially aggregated protein, the sample was first added to a solution of 14% PEG3350, 10% ethylene glycol, and 0.1 M HEPES, pH 7.0 in a ratio of 3:2 protein:buffer, and incubated at ~ 20 degC for 15 min. The solution was then centrifuged at 18,000 x g for 15 min. The supernatant was then applied to a coverslip in 3 microL hanging drops and placed over reservoir buffer containing 16% PEG3350, 10% ethylene glycol, and 0.1 M HEPES pH 7.0 and incubated at 20 °C. Large ~700 x 500 x 50 micrometer plate crystals 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.