plasmodium yoelii ribonucleotide reductase small subunit

Target ID:

PY03671

Deposition Date:

Monday 5th March 2007

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2P1I

Authors:

A.K.WERNIMONT,A.DONG,J.CHOE,M.GAO,J.WALKER,J.LEW,Z.ALAM,Y.ZHAO,P.NORDLUND,C.H.ARROWSMITH,A.M.EDWARDS,J.WEIGELT,M.SUNDSTROM,A.BOCHKAREV,R.HUI,J.D.ARTZ,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

2P1I

tabs

Structure Details

Ribonucleotide reductases (RNRs) catalyze the reduction of all four main ribonucleotides to their corresponding deoxyribonucleotides. As such, these enzymes are critical to the survival of most organisms. They are responsible for the proper maintenance of the pool of DNA precursors.
RNRs are regulated by ATP (activation) and dATP (feedback inhibition); but they are also regulated by substrate specificity for the triphosphate nucleotides such that a balance of the essential nucleotides can be maintained during DNA synthesis. Since the key role of RNRs is DNA synthesis, they have been attractive with respect to anti-bacterial, anti-viral, and anti-cancer drug development.

Eukaryotic RNRs are comprised of two subunits: (1) the R1 subunit that binds the substrate and contains an allosteric effector binding site; and (2) the R2 subunit that contains the di-iron tryosyl radical cofactor essential for catalysis. In most eukaryotes and some prokaryotes, the active RNR enzyme is a tetrameric complex made up of homodimers of both subunits.

In budding yeast, two distinct R1 genes (dubbed RNR1 and RNR3) as well as two distinct R2 genes (RNR2 and RNR4) have been found. In Plasmodium falciparum, the presence of two copies of the R2 gene, dubbed PfR2 (PlasmoDB ID: PF14_0053) and PfR4 (PlasmoDB ID: PF10_0154), has previously been found by means of sequence analysis of a digested genomic DNA library. Furthermore, homology analysis confirms both genes are present in P. vivax, P. yoelii, P. berghei, P. chabaudi and P. knowlesi, as well as C. parvum.

We present here the structure of RNR-R2 from the rodent malaria pathogen Plasmodium yoelii. We have also previously solved

the structure
of RNR-R2 from P. vivax.

Materials and Methods

Py-RNR-R2: Plasmodium yoelii ribonucleotide reductase, R2 subunit (PY03671)

PDB:2P1I

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PY03671
Entry Clone Source:Plasmodium yoelii genomic DNA
SGC Clone Accession:plate MAC00E:A5
Tag:N-terminal: N-terminal His-tag with integrated TEV protease site (*): MGSSHHHHHHSSGRENLYFQ*G
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqgSKSASFSKQEKEFSDFQKSKESNEKILNKESNRFTLHPIMYPEVWNFYKKAEASFWTAEEIDLSSDLKDFEKLNVNEKHFIKHVLAFFAASDGIVLENLASKFLREVEIIEAKKFYSFQIAVENIHSETYSLLIDNYIKDEKERLNLFHAIENIPAIKNKALWAAKWINDTNSFAERIVANACVEGILFSGSFCAIFWFKKQNKLHGLTFSNELISRDEGLHTDFNCLIYSLLENKLPENVVQNIVKEAVEVERSFICESLPCDLIGMNSRLMSQYIEFVADRLLECLGCSKVFHSKNPFNWMDLISLQGKTNFFEKRVADYQKSGVMAQRKEQVF
Vector:p15-Tev-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 2.5, 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 3 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 protein was dialysed overnight in a dialysis cassette (Pierce) in Crystal Buffer. The following day, it was concentrated using a 15 mL Amicon Ultra centrifugal filter device Millipore (5 kD cutoff).Protein concentration was estimated by means of absorbance at OD280. Aliquots of the purified protein were flash frozen in N2(l) and stored at -80°C.

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:
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of sitting drop vapor diffusion in Intelli-Plate. The plate was set with 0.5 microL protein and 0.5 microL buffer in each drop, and 100 microL reservoir volume per well. Crystals grew in 2.0 M sodium formate, 100 mM Hepes and pH 8.0 at 20 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Ekland E, Uhlin U, Farnegardh M, Logan DT, Nordlund P. Review: Structure and function of the radical enzyme ribonucleotide reductase. Progress Biophys. Mol. Biol., 77 (2001) 177-268.
Bracchi-Ricard V, Moe D, Chakrabarti D. Two Plasmodium falciparum Ribonucleotide Reductase Small Subunits, PfR2 and PfR4, Interact with Each Other and are Components of the in vivo Enzyme Complex. J. Mol. Bio. (2005) 347, pp. 749-758.