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Cryptosporidium parvum farnesyl diphosphate synthase with zoledronate bound

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PDB Code 2HER Target Class Malaria

Target cgd4_2550
Alias n/a
Disease Area/Function parasitic disease
Date Deposited 2006-06-21
Authors M.CHRUSZCZ, J.ARTZ, H.ZHENG, A.DONG, J.DUNFORD, J.LEW, Y.ZHAO, I.KOZIERADSKI, K.L.KAVANAUGH, U.OPPERMAN, M.SUNDSTROM, J.WEIGELT, A.EDWARDS, C.ARROWSMITH, A.BOCHKAREV, R.HUI, W.MINOR, STRUCTURAL GENOMICS CONSORTIUM (SGC)
Related Structure 2O1O

Struc Details Tabs

Structure Details
Isoprenoids such as sterols and ubiquinones are a diverse and universal family of natural products. Their biosynthesis typically involves condensation of different numbers of isopentenyl diphosphate units (IPP).1 In mammals, IPP is produced by means of the mevalonate pathway with eponymous mevalonic acid as the starting metabolite. Simpler organisms, including eubacteria, algae and some plants, undergo an alternative non-mevalonate mechanism involving conversion of glyceraldehyde 3-phosphate (G3P) and pyruvate to 1-deoxy-D-xylulose 5-phosphate (DOXP) and subsequently to 2-C-methyl-D-erythritol 4-phosphate (MEP), giving rise to the DOXP pathway or the MEP pathway.

With studies uncovering the ineffectiveness of inhibitors of the mevalonate pathway, identification of enzymes such as DOXP synthase and DOXP reductoisomerase, as well as effectiveness of fosmidomycin in curing malaria in mice,2 it has been established that Plasmodium parasites produce IPP by means of the MEP pathway. Specifically, it has been shown that this pathway takes place inside the apicoplast organelle in both Plasmodium and Toxoplasma parasites.

Cryptosporidium parvum is an obligate parasite responsible for cryptosporidiosis in farm animals and humans. Genome sequencing and annotation has confirmed the absence of the apicoplast organelle in Cryptosporidium and apicoplast-localized pathways. At the same time, these parasites also do not appear to have the key enzymes needed to undergo the mevalonate pathway. Beyond this, the isoprenoid pathway in the simpler Apicomplexan organisms Cryptosporidium parvum and hominis is under-studied and remains essentially unknown.

Bisphosphates are drugs that inhibit the resorption of bone. The molecular target of nitrogen-containing bisphosphonates (N-BP) is thought to be FDPS, because these drugs have been shown to inhibit the mevalonate pathway in osteoclasts and macrophages. In support, crystal structures of hFDPS with N-BP have recently been solved.

The C. parvum genome encodes a gene (cgd4-2550) which is less than 30% identical to Plasmodium and human FPPS. We have dubbed this enzyme polyprenyl pyrophosphate synthase (PPPPS) and have solved its structure in complex with the nitrogen-containing bisphosphonate zoledronate.

See also

References

  1. Kavanagh KL, Dunford JE, Bunkoczi G, Russell RG, Oppermann U. The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding. J Biol Chem. 2006.
  2. Kavanagh KL, Guo K, Dunford JE, Wu X, Knapp S, Ebetino FH, Rogers MJ, Russell RG, Oppermann U. The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs. Proc Natl Acad Sci U S A., 103(20):7829-34 (2006).
  3. Rondeau, J.-M., Bitsch, F., Bourgier, E., Geiser, M., Hemmig, R., Kroemer, M., Lehmann, S., Ramage, P., Rieffel, S., Strauss, A., Green, J.R., Jahnke, W. Structural Basis for the Exceptional in vivo Efficacy of Bisphosphonate Drugs ChemMedChem v1 pp.267-273, 2006.
  4. Abrahamsen MS, Templeton TJ, Enomoto, S, Abrahante, JE, Zhu, G, Lancto, CA, Deng, M, Liu, C, Widmer, G, Tzipori, Z, et al. The complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304: 441–445, 2004.
Materials & Methods
StructureCp-FDPS-Zoledronate
PDB Code2HER
Entry clone accession cgd4_2550 (www.cryptodb.org)
Entry clone source Cryptosporidium parvum Iowa strain gDNA
SGC clone accession cgd4_2550:E38-L384; MAC017:C2
Tag N-terminal: His6-tag with integrated TEV protease site: mgsshhhhhhssgrenlyfq*g
Construct sequencemgsshhhhhhssgrenlyfqgEYDYTDFINYYDKFKVIVYNVLKKLPLNDEIRKPVIEYYLNCIDYNVKKGKHIRGKILV
LISSLSSAYSNIKRDSIYLLGWVVEAIQALILIADDIMDSGKFRRGAPCWYIVHGQSNAINDIFFLKMLSLSLIFELSSV
FGNDIVMKIQKIYNESIFFTVLGQHLDLSYFDLSKADKISERYFSMVEMKTSRYTFYMPVFFGLTLSEIQVSSAQLNLIE
AILYKLGEFYQVHNDVSDYLFNDSNADDICRFKLTWPLQKSFEIADEEMKLKISENYGKNSSLVKDCYNLLKINEHYLEY
QRNALDYLIKLVKDITDDSLQKVFIHLIHQISELITNSRSNADSNNSL
Vector p15-tev-lic
Expression host E. coli BL21-(DE3)-R3
Growth method Cp-FDPS was expressed in E. coli BL21-(DE3)-Rosetta-Oxford cells in Terrific Broth (TB) in the presence of ampicillin/chloramphenicol (50 μg/mL and 25 μg/mL respectively). A single colony was inoculated into 10 mL of LB with of ampicillin/chloramphenicol (50 μg/mL and 25 μg/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37 ºC. The culture was transferred into 50 mL of TB with 50 μg/mL ampicillin in a 250 mL shaking flask and incubated at 37 ºC for 3 hours. Then the culture was transfer into 1.8 L of TB with 50 μg/mL kanamycin 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 ºC, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 ºC.
Extraction procedure The culture was harvested by centrifugation. Pellets from 4 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5 % glycerol) with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 ºC were thawed overnight at 4 ºC 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 a Beckman JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 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 - 2.5 mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer 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. Each 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 - 2.5 mL/min.
After washing, the protein was eluted with 15 mL of Elution Buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, and 5 % glycerol). EDTA was immediately added to the elution fraction to 1 mM; and DTT was added to 1-5 mM after approximately 15 more minutes.
The Ni-NTA purified protein was loaded onto a 26/60 S200 Superdex gel filtration column; and the major peak corresponding to the Cp-FDPS dimer was collected and concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). The concentrated protein was stored at 4 ºC.

Stock concentration - 7.1 mg/mL.

Crystallization Purified Cp-FDPS was crystallized using the hanging drop vapor diffusion method in a VDXm plate with 350 µL of mother liquor at 18 ºC. 1.5 µL of the protein solution (containing 10 mM Zoledronate, 10 mM isopentenyl pyrophosphate, and 10 mM MgCl2) was mixed with 1.5 µL of the reservoir solution (containing 1.6 M ammonium sulfate, 10 mM MgCl2, and 100 mM HEPES, pH 7.5). Crystals appeared after 4-5 weeks.
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