Leishmania Major HSP90, middle and C-terminal domain

Target ID:

PP-HSP90

Deposition Date:

Thursday 21st May 2009

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

3HJC

Authors:

Wernimont AK, Tempel W, Lin YH, Hutchinson A, Mackenzie F, Fairlamb A, Kozieradzki I, Cossar D, Zhao Y, Schapira M, Bochkarev A, Arrosmith CH, Bountra C, Weigelt J, Edwards AM, Ferguson MAJ, Hui R, Pizarro JC, Hills T

Site:

Toronto

3HJC

tabs

Structure Details

The molecular chaperone Hsp90 (heat shock protein 90) is up-regulated under stress conditions and has been considered the Achilles heel of cells undergoing rapid replication making it a valuable drug target in cancer and infectious diseases.

Hsp90 is composed of three distinct domains, an N-terminal nucleotide binding domain followed by a middle and C-terminal domain. The middle domain is connected to the N-terminal domain via an acid linker and these domains are responsible for the binding of the protein substrates of the Hsp90, called clients in this context. The binding of the clients and their further activation by the chaperone are coupled to the catalytic ATPase function of the Hsp90. The proposed mechanism involves a transient association of the N-terminal domains with a condensation of the Hsp90 dimer. The C-terminal domain is responsible for the dimerization of the chaperone in the absence of clients or ATP. In addition to the structural function, the C-terminal domain has been implicated in the binding of several antibiotics known to inhibit Hsp90 activity, e.g. Coumermycin and Novobiocin. The inhibitors that bind to this domain of the chaperone are less well characterized compared to the ones binding the N-terminal domain, in addition there is no structural knowledge concerning their interaction with the Hsp90. To gain further understanding in the binding functions of the C-terminal domain, and to establish the foundations to explore the inhibitory potential of it, we have determined the structure of the middle and C-terminal domains of Leishmania major Hsp90. The reported apo structure may be considered the resting/inactive form of the full length molecule and increases our understanding of the function of the enzyme since it is one of the few middle plus C-terminal domains ever crystallized of an Hsp90. This structure would help us rationalizing the binding information of the C-terminal inhibitors of the chaperone and constitutes an essential piece of information for the development of chemical probes aimed to explore its function and possible inhibition consequences in L. major.

Materials and Methods

LmHSP90

PDB:3HJC

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:LmjF33.0312:H265-V690:D6
Tag:mhhhhhhssgrenlyfqg
Host:BL21-(DE3)-V2R-pRare2.

Construct

Prelude:
Sequence:HKPLWTRDPKDVTKEEYAAFYKAISNDWEDPAATKHFSVEGQLEFRSIMFVPKRAPFDMFEPNKKRNNIKLYVRRVFIMDNCEDLCPDWLGFVKGVVDSEDLPLNISRENLQQNKILKVIRKNIVKKCLEMFDEVAENKEDYKQFYEQFGKNIKLGIHEDTANRKKLMELLRFYSTESGEEMTTLKDYVTRMKAGQKSIYYITGDSKKKLETSPFIEQARRRGLEVLFMTEPIDEYVMQQVKDFEDKKFACLTKEGVHFEESEEEKQQREEEKAACEKLCKTMKEVLGDKVEKVIVSERLSTSPCILVTSEFGWSAHMEQIMRNQALRDSSMAQYMMSKKTMELNPRHPIIKELRRRVGADENDKAVKDLVFLLFDTSLLTSGFQLEDPTGYAERINRMIKLGLSLDEEEEEAAEAPVAETAPAEV
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:Leishmania major PP-HSP90(LmjF33.0312) 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 15degC, 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 PP-HSP90 (LmjF33.0312).

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 sonicated for 10 minutes 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: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 (LmjF33.0312) was concentrated to 28.6 mg/ml and stored at 4C. The protein was diluted to 15 mg/ml before use in crystal trials.
Ligand
MassSpec:
Crystallization:The protein was crystallized in 2.5 M Ammonium sulfate, 0.1 M Sodium acetate pH 4.6 at 20degC in using the sitting drop method.
NMR Spectroscopy:
Data Collection:
Data Processing: