Plasmodium falciparum putative activator of Hsp90 ATPase (PFC0270w)

Target ID:

PP-HSP90

Deposition Date:

Wednesday 26th May 2010

Families:

Miscellaneous

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

3N72

Authors:

Wernimont AK, Dong A, Hutchinson A, Sullivan H, MacKenzie F, Kozieradzki I, Cossar D, Bochkarev A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Hui R, Pizarro JC, Hills T

Site:

Toronto

3N72

tabs

Structure Details

The Heat shock protein 90 (Hsp90) is a widespread molecular chaperone, assisting in the maturation process of many proteins involved in cellular signaling and cell cycle regulation. Its chaperone function is dependent upon a series of conformational changes and domain rearrangements tied to an enzymatic ATPase activity. Additionally, a cohort of co-chaperones assist Hsp90 to fulfill its cellular role with several among them modulating its enzymatic activity. One of such is AHA1 (Activator of Hsp90 ATPase homolog 1), first isolated in Saccharomyces cerevisiae and the only known enhancer of the molecular chaperone's ATPase activity. In humans and yeast, AHA1 is composed of two domains, both required for tight binding to the chaperone. The N-terminal domain is typically referred to as the AHA1N domain and known to interact with the middle domain of Hsp90. The corresponding yeast complex has been previously captured in a crystallographic structure (PDB ID: 1USU, Meyer et al, 2004). The C-terminal domain, known as the AHSA1 domain, also contributes by interacting with the N-terminal domain of Hsp90 (Retzlaff et al, 2010).

Plasmodium parasites feature a somewhat different co-chaperone system. The gene PFC0270w encodes a protein with two AHA1N-like domains, which we dub PfAHA-1N-N and PfAHA1N-C. In addition, the AHSA1 homologue is encoded by an independent gene, namely PFC0360w. We have solved the structure of PfAHA1N-N. It is highly similar to the AHA1-N domain of yeast (PDB ID: 1USU).

Materials and Methods

Aha-1

PDB:3N72

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:PFC0270w:E15-K160:D06
Tag:mhhhhhhssgrenlyfqg
Host:BL21-(DE3)-V2R-pRare2.

Construct

Prelude:
Sequence:ERNYNKWAESYIKYNLSNLKIEKEDLTIYFDNLQVSGNACVSIRKGKQINSFEYIIKFEWLYSKKKEGKDYFGGSVEIPDFSTFSLEENDYAINIERTDESENLRFIYDSILKKEGKEKIKECLKNFQEDLLKHDKNESNKELKIK
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:Plasmodium falciparum Aha1, PFC0270w, 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 34microgram/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37degC. 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°C, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15degC

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 PFC0270w.

The sample was then loaded onto a superdex 75 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-AHA1 (PFC0270w) was concentrated to16.4 mg/ml and stored at 4C. The protein was diluted to 6 mg/ml (0.3 mM) prior to crystallization.

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 2 L of culture were 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)). 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 a Beckman JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 degC.
Concentration:
Ligand
PFC0270w was crystallized in the presence of 0.24 mM PF07_0029: N310-E720:B4MassSpec:
Crystallization:Crystallization plate: IC-MAZ3FL:F8-2
buffer: 30% PEG 2000 MME, 0.1 M potassium thiocyanate at 20 degC
Cryo condition: crystallization condition +15% ethylene glycol
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Meyer P, Prodromou C, Liao C, Hu B, Roe SM, Vaughan CK, Vlasic I, Panaretou B, Piper PW, Pearl LH. EMBO J. 2004 Mar 24;23(6):1402-10.
Retzlaff M, Hagn F, Mitschke L, Hessling M, Gugel F, Kessler H, Richter K, Buchner J. Mol Cell. 2010 Feb 12;37(3):344-54.