Plasmodium falciparum HSP90, N-terminal domain, in complex with ADP analog

Target ID:

PP-HSP90

Deposition Date:

Wednesday 22nd July 2009

Families:

Miscellaneous

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

3IED

Authors:

Pizarro JC, Wernimont AK, Lew J, Hutchinson A, Artz JD, Amaya MF, Plotnikova O, Vedadi M, Kozieradzki I, Weigelt J, Edwards AM, Arrowsmith CH, Bountra C, Botchkarev A, Hui R, Hills T

Site:

Toronto

ICM Datapack:

ICM Datapack

3IED

tabs

Structure Details

Heat shock protein 90 (Hsp90), named after its apparent molecular weight, is a molecular chaperone whose functions include assisting protein folding and maturation. Under normal conditions it accounts for 1-2 % of the total cell proteome but it could be increased under stress situations. This essential chaperone is composed of three distinct domains, an N-terminal ATP binding domain, a middle domain and a C-terminal domain involved in substrate binding and dimerization. The Hsp90 activities are coupled with ATP hydrolysis that triggers conformational changes, including a transient association of the N-terminal domains. The relationship between the ATPase and chaperone activities is not fully understood in part due to the fact that several accessory proteins, such as AHA1, cdc37 and P23, could modify the Hsp90 activity . However, it has been shown that interfering with the ATPase activity, either by mutation or pharmacological means, severely compromises Hsp90 functions.

Hsp90, despite being a molecular chaperone for several client proteins involved in cell signaling, is actively targeted in research of anticancer therapeutics mainly due to the low toxicity reported for the inhibitors that have reached the clinical stage. This specific inhibition of a rather promiscuous protein is poorly characterized but is known to be linked to the largest structural changes that are involved in the chaperone cycle. Most of the Hsp90 inhibitors target the ATP binding site in the N-terminal domain, highlighting the unique structural properties of this protein and the paradoxical behavior of this target. In the case of infectious diseases, Hsp90 has a dual role making it also a very attractive drug target. On one hand, pathogens have co-opted this host chaperone to assist the folding/maturation of their own proteins hence inhibiting the host Hsp90 will have a deleterious effect on the infectious agent. On the other hand, its contribution to survival under drug exposure is directly linked to Hsp90's involvement in stress responses and represents an important source of phenotypic resistance. Therefore, hampering the chaperone activity will reduce the pathogen's ability to combat the drug effects. Additionally, specific molecular probes for parasitic protozoan Hsp90's will help to characterize its function throughout the organism's life cycle.

In the case of Plasmodium falciparum, its genome contains at least four copies of Hsp90, PF07_0029, PF11_0188, PF14_0417 and PFL1070c. PF07_0029 and PF11_0188 being the most abundant Hsp90's and they are predicted to be the cytoplasmic and the plastid version of the chaperon, respectively. PLF1070c shows variable expression in the different stages of the parasite life cycle and its sequence is more related to the endoplasmins. Finally, PF14_0417 is a large protein of over a 100 kDa, presenting N and C terminal extensions specific to Plasmodial species. Previously, we have solved the structure of the middle domain of PF14_0417 (1Y6Z).

Here we report the structure of the N-terminal domain of P.falciparum Hsp90 (PF14_0417:S97-E347) in complex with AMPPN (AMP Phosphoramidate) to 2.0 Å resolution. This structure highlights a rather unique ATP binding site when compared with others Hsp90. It shows a hydrolyzed form of AMPPNP since the γ phosphate was absent from the electron density. We conclude that the substrate was hydrolyzed during the course of the crystallization experiment. This is the second structure in the PDB with a hydrolyzed AMPPNP, the other structure is a SERCA ATPase (3BA6).

Materials and Methods

HSP90

PDB:3IED

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PF14_0417
Entry Clone Source:
SGC Clone Accession:PF14_0417:S97-E347:E10
Tag:N-terminal tag: mhhhhhhssgrenlyfqg
Host:BL21-(DE3)-V2R-pRare2.

Construct

Prelude:
Sequence:SPVEKYNFKAEVNKVMDIIVNSLYTDKDVFLRELISNASDACDKKRIILENNKLIKDAEVVTNEEIKNETEKEKTENVNESTDKKENVEEEKNDIKKLIIKIKPDKEKKTLTITDNGIGMDKSELINNLGTIAQSGTAKFLKQIEEGKADSNLIGQFGVGFYSSFLVSNRVEVYTKKEDQIYRWSSDLKGSFSVNEIKKYDQEYDDIKGSGTKIILHLKEECDEYLEDYKLKELIKKYSEFIKFPIEIWSE
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:Plasmodium falciparum PP-HSP90(PF14_0417) was expressed in E. coli BL21(λDE3) V2R pRare2 in TB growth media in the presence of carbenicillin/chloramphenicol (100 microgram/mL and 34 microgram/mL, respectively). A single colony was inoculated into 25 mL of LB with of carbenicillin/chloramphenicol (100 microgram/mL and 34 microgram/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37 degC. Then the culture was transferred into 900 mls of TB with 100 microgram/mL Carbenicillin and 34 microgram/ml chloramphenicol , 0.3 mL of antifoam (Sigma), 9 mls of 0.83 M MgSO4 and trace elements in a 1L 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 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 2mL 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. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2 - 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer. 1 mM TCEP and 1 mM EDTA was added to the eluted PP-HSP90 (PF14_0417).

The sample was then loaded onto a superdex 200 gel filtration column. The eluted protein ( in 10 mM Hepes, pH 7.5 and 500 mM NaCl) was concentrated using a 15 ml Amicon Ultra centrifugal filter device (Millipore) with a 10 kDa cutoff. PP-HSP90 (PF14_0417) was concentrated to 33 mg/ml and stored at 4 degC. The protein was diluted to 15 mg/ml before use in crystal trials.

Extraction

Procedure
The culture was harvested by centrifugation. A pellet from 1 L of culture was resuspended to approximately 40 mL/L of cell culture in Binding Buffer with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). The resuspended pellet , stored at -80 degC, was thawed overnight at 4 degC on the day before purification. Prior to mechanical lysis, the pellet from 1 L of culture was pretreated with 0.5 % CHAPS and 500 units of benzonase and protease inhibitor (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)) 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 degC.
Concentration:33 mg/ml.
Ligand
MassSpec:
Crystallization:The protein was crystallized in 1.5 M NaCitrate, 0.1 M Na Cacodylate pH 5.5 at 20 degC using the sitting drop method. The ligand added at 2 mM AMPPNP, 4 mM MgCl2, 2 mM TCEP.
NMR Spectroscopy:
Data Collection:
Data Processing: