Cryptosporidium parvum cyclase-associated protein

Target ID:

Cp-PFA0260c

Deposition Date:

Wednesday 14th September 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2B0R

Authors:

W.TEMPEL,A.DONG,Y.ZHAO,J.LEW,I.KOZIERADZKI,Z.ALAM,M.MELONE,G.WASNEY,M.VEDADI,C.ARROWSMITH,A.EDWARDS,J.WEIGELT,M.SUNDSTROM,R.HUI,A.BOCHKAREV,J.ARTZ,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2B0R

tabs

Structure Details

Cyclase-associated proteins (CAP) are highly conserved G-actin-binding proteins
which participate in cell growth and cytoskeletal development,
including RNA localization and cell polarity establishment.
In the absence of CAP, change in cell shape often becomes noticeable,
along with accumulation of actin and mis-localized mRNA.

Despite their name, not all CAPs bind adenyl cyclase.
In yeast and humans, CAP comprises a N-terminal region that binds adenyl cyclase
and a C-terminal domain that binds monomeric G-actin, with a proline-rich,
SH3-binding region separating the two.1
In protozoans, CAP is typically mono-functional and defined only by the actin-binding domain.

Here, we present the 2.6 angstrom structure of Cp-CAP1
(cgd5_440),
cyclase-associated protein from Cryptosporidium parvym,
an apicomplexan organism and causative agent of cryptosporidiosis.
This protein is an ortholog of Pf-CAP1
(PFA0260c):
CAP1 from Plasmodium falciparum).

Similar to the C-terminal structures of CAP1 from humans
(1K8F)
and yeast,
Cp-CAP1 is a dimer with each monomer featuring a unique right-handed parallel Ã¢-helix
and the two monomers joined by wrapping their respective C-terminal Ã¢-hairpins around each other.
It is speculated that actin is bound in this region,
where there is a stretch of conserved residues E(x)nPEQ
with n = 3 or 4.
In Cp-CAP1, the sequence is ELAIPEQ.

Cp-CAP: Cryptosporidium parvum cyclase-associated protein

PDB:2B0R

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd5_440
Entry Clone Source:Cryptosporidium parvum strain Iowa genomic DNA
SGC Clone Accession:CP-PFA0260c; plate MR:H4
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPR*GS
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsKSQIYLKKEKKMKAARQVVTNGSPKVELQKDTYLVENHVNCADPITLSEGSIKNKVSVRCSQNSRIIVEQKVNSIFIENCVGCIFLVNGVISSIEIVNCDDIKLQMTGIVPTISLDKSNKVNIYTSKEGKNVEVYSSKSSEMNLLFPGEEEGDWKELAIPEQFVTKYNESKGKLESMVSPLYG
Vector:pET28a-LIC

Growth

Medium:Terrific Broth (TB)
Antibiotics:50 microG/mL kanamycin and 25 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 degC.

Purification

Procedure
The cleared cell lysate was loaded onto a DE52 (Whatman) column packed with 10 g of resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer), and subsequently onto a 2.5 mL Ni-NTA 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 at 2Â2.5 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 then added to 1 mM 15 minutes later.

The eluted protein was applied to a Sephadex S200 26/60 gel filtration column pre-equilibrated with Gel Filtration Buffer. The collected fractions corresponding to the eluted protein peak were concentrated using a 15 mL Amicon Ultra centrifugal filter device from Millipore (10 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:30 mg/mL for uncleaved protein and 16.9 mg/mL for cleaved protein
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of hanging drop vapor diffusion in Linbro plate. The plate was set with 1.5 microL cleaved protein and 1.5 microL buffer in each drop, and 300 microL reservoir volume per well. Crystals grew overnight in 17 % Peg 3350 and 200 mM diammonium tartrate at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Dodatko, T., Fedorov, A. A., Grynberg, M., Patskovsky, Y., Rozwarski, D. A., Jaroszewski, L., Aronoff-Spencer, E., Kondraskina, E., Irving, T., Godzik, A., Almo, S. C. (2004) Crystal Structure of the Actin Binding Domain of the Cyclase-Associated Protein Biochemistry 43: 10628-10641
Hubberstey, A. and Mottillo, E. (2002) Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization. FASEB J.: 487-499.