Cryptosporidium parvum cyclophilin (cgd2_4120)

Target ID:

cgd2_4120

Deposition Date:

Friday 20th April 2007

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2PLU

Authors:

A.K.WERNIMONT,J.LEW,T.HILLS,I.KOZIERADZKI,Y.H.LIN,A.HASSANALI,Y.ZHAO,M.SCHAPIRA,C.H.ARROWSMITH,A.M.EDWARDS,J.WEIGELT,M.SUNDSTROM,A.BOCHKAREV,R.HUI,J.D.ARTZ,T.XIAO,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

2PLU

tabs

Structure Details

Cyclosporin, also known as ciclosporin or cyclosporine, is an immunosuppressant drug originally identified from the fungus Tolypocladium inflatum Gams [1].
It is often prescribed to patients following organ transplant,
as well as for psoriassis and other auto-immune disorders.
Moreover, potent anti-parasitic activity [2] has been reported, including against malaria [3].
Concerns about adverse side-effects, however, limit the wide use of this drug for parasitic diseases.
There are over 20 variants of cyclosporin, all of which are cyclic undecapeptides.
Cyclosporin A (CsA) is best known, with other forms (e.g. B, C, D, E, G, H, etc.) varying
by individual residues or chiral deviations.

Cyclophilins are peptidyl-prolyl cis-trans isomerases (PPIase or PPI) named for their affinity for cyclosporin.
Prolyl isomerases (also PPIase) are a universally conserved family of enzymes
that converts the cis and trans isomers of peptide bonds with proline - an amino acid with a cyclic structure conferring unique conformational
rigidity to its peptide bond.
Consequently, unlkike other amino acids which preferentially form the trans peptide bond conformation,
both cis and trans isomers of proline are common.
Because proline isomerization is a rate-limiting step in protein folding, PPI's are considered chaperones.
Other PPIase groups including FK506-binding proteins (FKBP) and parvulins.

The Cryptosporidium parvum parasite is predicted to have 6 cyclophilin type PPI's encoded in its genome - cgd1_870, cgd2_4120, cgd2_1660, cgd7_520, cgd8_1560 and cgd8_2350,
in addition to two putative FK506-binding proteins - cgd6_2690 and cgd7_210.
Specifically, we have obtained two crystallographic structures of the Cryptosporidium parvum cyclophilin
encoded by the gene cgd2_4120 - the apo form
(2PLU)
and the second with cyclosporin A bound
(2POY).
Like other cyclophilins, our structures do not deviate from the canonical cyclophilin fold of a barrel formed by two sets of four anti-parallel beta strands, flanked spatially (but not sequentially) by two to three helices.
The conserved CsA-interacting residues are

R78, F83, M84, Q86, G95, A124, N125, A126, Q134, F136, W145 and L146,
specifically residues 1, 2, 3, 9, 10, 11 of CsA.
The other CsA residues (4, 5, 6, 7, 8) form a composite "effector" surface with other residues on the enzyme to interact with calcineurin.
As found in the past, binding of cyclosporin does not change the structure of the enzyme itself.

Also found on the Cp cyclophilin is a

protease-like catalytic triad - H115, S122, D146.
Its functional role has yet to be determined.

Cp-CyP: Cryptosporidium parvum cyclophillin (cgd2_4120)

PDB:2PLU

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd2_4120
Entry Clone Source:Cryptosporidium parvum strain Iowa genomic DNA
SGC Clone Accession:cgd2_4120:N3-V170; plate MAC01Y:E8
Tag:N-terminal His6-tag with integrated TEV cleavage site (*): mhhhhhhssgrenlyfq*g
Host:E. coli BL21-(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:gNPVVYFDISIGQTPAGRITMELFADKVPITAENFRALCTGEKGMGQSGKPLCYTGSFFHRIIPQFMIQGGDFTRGDGTGGESIYGSKFRDENFVYTHDAPFLLSMANAGPNTNGSQFFITTVPCPWLDGKHVVFGKVLEGMEVVKSIEKCGSQNGKPTKSVCITASGV
Vector:pET15-MHL

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 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 1.0-2.5 mL Ni-NTA (Qiagen) column at approximately 1-1.5 mL/min. The volume of the Ni-NTA resin was pre-determined by the predicted protein yield from test expression analysis. After the lysate was loaded, the DE52 was further washed with 20 mL of Binding Buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2 Â 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer. TCEP was added to 1-5 mM after approximately 15 more minutes.

The eluted protein from Ni-NTA was loaded onto a Sephadex S75 26/60 column equilibrated with Gel Filtration Buffer. The fractions from the peak eluting at the volume corresponding to monomeric protein were pooled and concentrated using a 15 mL Amicon Ultra centrifugal filter device from Millipore (10 kD cutoff). The protein sample identity and purity were evalulated by mass spectroscopy and 10% SDS-PAGE gel. The concentrated protein was flash frozen in liquid nitrogen and then stored at -80 degC.

Extraction

Procedure
Cells 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:
Ligand
None for 2PLUCsA for 2POYMassSpec:
Crystallization:The native protein was crystallized by means by sitting drop vapor diffusion in a 96-well Intelli-Plate. The plate was set with 0.5 microL protein and 0.5 microL buffer in each drop, and 350 microL reservoir volume per well. Crystals grew in 20% PEG 8000, 0.2M ammonium sulfate 0.1M sodium cacodylate, pH 5.5 at 18 degC.
For the CyP-CsA complex, cyclosporin A was added after gel filtration of the native protein at a ratio of 1:2. The mixture was then incubated at 4 degC under gental rocking overnight. After high speed centrifugation, the protein was concentrated for crystallization.Crystals formed in 25% w/v PEG 3350, 0.1M citric acid, pH 3.5 at 20 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Borel, JF. History of the discovery of cyclosporin and of its early pharmacological development. Wien Klin Wochenschr (2002) 114/12: 433â€“437.
High KP and Handschumacher RE. Immunity,
microbial pathogens and immunophilins: Finding
the keys, now where are the locks? (1995) Infect. Agents
Dis. 1, 121-135.
Nickell SP, Scheibel LW and Cole GA. Inhibition by cyclosporin A of rodent malaria in vivo
and human malaria in vitro (1982). Infect. Immun. 37, 1093-1100.
Peterson MR, Hall DR, Berriman M, Nunes, JA, Leonard GA, Fairlamb AH
and Hunter WN.
Three-dimensional Structure of a Plasmodium
falciparum Cyclophilin in Complex with the Potent
Anti-malarial Cyclosporin A.
J. Mol. Biol. (2000) 298, 123-133.