Plasmodium falciparum ATP-dependent ClpP protease, putative

Target ID:

PFC0310c

Deposition Date:

Tuesday 29th November 2005

Families:

Malaria

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

2F6I

Authors:

A.MULICHAK,P.LOPPNAU,J.BRAY,M.AMANI,M.VEDADI,G.WASNEY,P.FINERTY,M.SUNDSTROM,J.WEIGELT,A.EDWARDS,C.ARROWSMITH,R.HUI,O.PLOTNIKOVA,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2F6I

tabs

Structure Details

In many organisms, protein degradation is mostly performed by large protease complexes using energy-dependent mechanisms,
including the proteasome, Lon protease and the caseinolytic protease (Clp) family.1
Specifically, Clp proteases (clan SK and family S14 in the
MEROPS database)
are ATPase-peptidase assemblies which cleave substrates
such as casein and albumin.
The peptidase subunit, which may for example be ClpP or ClpQ, is a serine protease which forms dual oligomeric rings.
ATP-dependent chaperones, including for instance ClpA, ClpC or ClpX and also oligomeric,
regulate entry of substrate proteins at one or both ends.
In the absence of an ATPase, Clp proteolytic subunits are only weakly active against small peptides.

Although ClpP is not generally considered essential,
a novel antibiotic mechanism of action has been discovered based on de-regulating ClpP
and unleashing its proteolytic activity in the absence of ATPase interaction.2

The Clp family can be found in the Plasmodium falciparum genome,
including Pf-ClpP (PFC0310c).
Pf-ClpP is a multimeric serine protease with the catalytic triad S264-H289-D338
housed in the C-terminal domain and a large N-terminal region
without a conserved domain, presumably forming a large ATPase-interacting region.

Clp proteases are found in the cytoplasm of bacteria and organellar compartments in eukaryotes.
For instance, human ClpP is localized in mitochondria while in arabidopsis,
the mitochondria and chloroplast are home to this class of proteases.
Pf-ClpP is targeted for the apicoplast,
presumably responsible for degradation of denatured proteins in this vestigial plastid.

Our Pf-ClpP structure spans the C-terminal catalyic domain (D179-K370),
which is over 40% identical to both E. coli ClpP
(1TYF).3
and the N-terminal catalytic domain of human ClpP
(1TG6).4 Â

ClpP monomer with catalytic triad highlighted in red

Like the E. coli and human orthologs, Pf-ClpP features dual heptamers.
In the so-called handle region,3,4,5
each monomer contains a disordered region (to varying degrees) near the catalytic histidine
(circularly highlighted to the right),
extending to the alpha helix which, in the E. coli and human structures,
has been dubbed helix E and is the intercalation site of the two rings.
Therefore, it remains to be confirmed whether the Pf-Clp heptamers
stack the same way as in previous structures.

Materials and Methods

Pf-Clp: Plasmodium falciparum ClpP protease

PDB:2F6I

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PFC0310c
Entry Clone Source:Plasmodium falciparum 3D7 gDNA
SGC Clone Accession:PFC0310c:D179-K370; plate MAC2004:B3
Tag:N-terminal His-tag with integrated Thrombin protease site: MHHHHHHSSGVDLGTENLYFQ*sm
Host:E. coli BL21-(DE3)-CodonPlus-RIL from Stratagene

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmDIKDMKKDVKLFFFKKRIIYLTDEINKKTADELISQLLYLDNINHNDIKIYINSPGGSINEGLAILDIFNYIKSDIQTISFGLVASMASVILASGKKGKRKSLPNCRIMIHQPLGNAFGHPQDIEIQTKEILYLKKLLYHYLSSFTNQTVETIEKDSDRDYYMNALEAKQYGIIDEVIETKLPHPYFNKVEK
Vector:pET21a-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, both columns were washed with 20 mL of Binding Buffer. 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:30 mg/mL
Ligand
MassSpec:
Crystallization:The protein was crystallized by means of hanging drop vapor diffusion in a 24-well Linbro Plate. The plate was set with 1.5 microL protein and 1.5 microL buffer in each drop, and 500 microL reservoir volume per well. Crystals grew in 100 mM cacodylate buffer, pH 7.0, 200 mM ammonium sulfate and 23% PEG MME 550 at 18 degC.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Smith, C., Baker, T. and Sauer, R. Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Biochemistry 96 (12), 6678-6682, (1999).

2. Brotz-Oesterhelt, H., Beyer, D., Kroll, H., Endermann, R., Ladel, C., Schroeder, W., Hinzen, B., Raddatz, S.,
Paulsen, H., Henninger, K., Bandow, J., Sahl, H. and Labischinski, H. Dysregulation of bacterial proteolytic machinery
by a new class of antibiotics. Nature Medicine, 11 (10) 1082-1087 (2005).

3. Wang, J., Hartling, J. and Flanagan, J. The Structure of ClpP at 2.3 AÂ° Resolution Suggests a Model for ATP-Dependent Proteolysis. Cell, Vol. 91, 447â€“456, 1997.

4. Kanga, S., Maurizia, M., Thompson, M., Mueserb, T. and Ahvazib, B. Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP. Journal of Structural Biology 148, 338â€“352, 2004.

5. Gribun, A., Kimber, M. S., Ching, R., Spranger, R., Fiebig, K. M., & Houry, W. A. The ClpP Double-Ring Tetradecameric Protease Exhibits Plastic Ring-Ring Interactions and the N-Termini of Its Subunits Form Flexible Loops that are Essential for ClpXP and ClpAP Complex Formation. Journal of Biological Chemistry 280(16), 16185-16196 (2005).