Plasmodium falciparum putative activator of Hsp90 ATPase homolog 1-like protein (PFC0360w)

Target ID:

PP-AHA1

Deposition Date:

Tuesday 15th June 2010

Families:

Miscellaneous

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

3NI8

Authors:

Wernimont AK, 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

3NI8

tabs

Structure Details

The Heat shock protein 90 (Hsp90) is a widespread molecular chaperone, assisting 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 PfAHSA1. It is highly similar to the human AHSA1 structure (PDB ID: 1X53).

Materials and Methods

Aha-1

PDB:3NI8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:PFC0360w:M1-K140:A8
Tag:mhhhhhhssgrenlyfqg
Host:BL21-(DE3)-V2R-pRare2.

Construct

Prelude:
Sequence:MSFEITEEYYVPPEVLFNAFTDAYTLTRLSRGSLAEVDLKVGGKFSLFSGSILGEFTEITKPHKIVEKWKFRDWNECDYSTVTVEFISVKENHTKLKLTHNNIPASNKYNEGGVLERCKNGWTQNFLHNIEVILGYPKKK
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:Plasmodium falciparum Aha1, PFC0360w, 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 37°C. 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 157°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 PFC0360w.

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 (PFC0360w) was concentrated to 24.6 mg/ml and stored at 4C. The protein was diluted to 12.3 mg/ml (0.67 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
MassSpec:
Crystallization:Crystallization plate: IC-MAZ3M6:H12-2buffers: 20% isopropanol, 20% PEG 4K, 0.1M Sodium Citrate pH 5.6 at 20 degCCryo conditions: crystals were pulled through Al\'s oil + 20% reservoir layered over top of crystallization drop
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.