Human Paraplegin, AAA domain in complex with ADP

Target ID:

SPG7A

Aliases:

CARCMARFLJ37308MGC126331MGC126332PGNSPG5CSPG7

Deposition Date:

Thursday 16th August 2007

Families:

ATPases

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

2QZ4

Authors:

T.KARLBERG,L.LEHTIO,C.H.ARROWSMITH,H.BERGLUND,R.D.BUSAM, R.COLLINS,L.G.DAHLGREN,A.EDWARDS,S.FLODIN,A.FLORES, S.GRASLUND,M.HAMMARSTROM,M.D.HERMAN,I.JOHANSSON,A.KALLAS, T.KOTENYOVA,M.MOCHE,M.E.NILSSON,P.NORDLUND,T.NYMAN,C.PERSSON, J.SAGEMARK,M.SUNDSTROM,L.SVENSSON,A.G.THORSELL,L.TRESAUGES, S.VAN DEN BERG,J.WEIGELT,M.WELIN,L.HOLMBERG-SCHIAVONE

Site:

Stockholm

ICM Datapack:

ICM Datapack

2QZ4

tabs

Structure Details

Human Paraplegin (SPG7) is an ATP-dependent m-AAA protease in mitochondria and is specifically shown to be involved in ribosome maturation. Mutations in the spg7 gene cause axonal degeneration in hereditary spastic paraplegia (HSP). The disease is most commonly manifested in spasticity and weakness of the lower limbs.

Paraplegin belongs to the AAA+ superfamily (ATPases associated with a variety of cellular activities) and consists of three identifiable domains: an extracellular FtsH-domain which is found in membrane-bound ATP-dependent proteases, an AAA-domain and a peptidase M41 domain, which is a metalloprotease domain. The protein is located to the inner mitochondrial membrane.

Here, the crystal structure of the AAA-domain of human Paraplegin is presented in complex with the nucleotide ADP at 2.2 Ã… resolution. The overall structure is comprised of a five-stranded parallel βâ€“sheet flanked by αâ€“helices, that together with a four-helix bundle is forming the nucleotide binding pocket. The nucleotide ADP was clearly seen in the electron density, however no magnesium was observed despite high concentrations in purification and crystallization conditions. The βâ€“phosphate of ADP was seen bound to a conserved P-loop motif (GPPGCGKT). In bacteria a conserved orthologue, the ATP-dependent metalloprotease FtsH has been structurally determined (the AAA-domain and the protease domain). The overall architecture is that of a hexamer. The presented structure of the AAA-domain of human Paraplegin alone is however monomeric.

Materials and Methods

SPG7

PDB:2QZ4

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|62020635
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Host:E.coli BL21-Gold(DE3)pRARE2, where BL21-Gold(DE3) cells (Stratagene) have been transformed with pRARE2 originating from the Rosetta2 strain (Novagen). The pRARE2 plasmid supplies tRNAs for rare codons.

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smGVSFKDVAGMHEAKLEVREFVDYLKSPERFLQLGAKVPKGALLLGPPGCGKTLLAKAVATEAQVPFLAMAGAEFVEVIGGLGAARVRSLFKEARARAPCIVYIDEIDAVGKKRSTTMSGFSNTEEEQTLNQLLVEMDGMGTTDHVIVLASTNRADILDGALMRPGRLDRHVFIDLPTLQERREIFEQHLKSLKLTQSSTFYSQRLAELTPGFSGADIANICNEAALHAAREGHTSVHTLNFEYAVERVLAGTAKKSKILSK
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Freshly transformed cells were used to inoculate 40 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 37 ºC overnight. The overnight culture (40 ml) was used to inoculate 2 x 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin, 34 µg/ml chloramphenicol and approximately 500 µl BREOX FMT 30 anti-foam solution (Cognis Performance Chemicals UK Ltd). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~1.3. The culture was down-tempered to 18 ºC over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,700 x g, 10 min, 4 ºC). The resulting cell pellet (64.2 g wet cell weight) was resuspended in lysis buffer (2 ml/g cell pellet) supplemented with two tablets of Complete EDTA-free protease inhibitor (Roche Applied Science) and stored at -80 ºC.

Purification

Procedure

Columns
IMAC: Ni-charged 1 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 200 Prep Grade (GE Healthcare)

Procedure
Purification of the protein was performed on an ÄKTAprime system (GE Healthcare). The filtered lysate was divided into two halves and loaded onto two separate HiTrap Chelating columns, pre-equilibrated with IMAC wash1 buffer. The columns were washed with IMAC wash1 buffer followed by IMAC wash2 buffer and the protein was eluted with IMAC elution buffer. Due to precipitation, the samples were diluted in GF-buffer and 1-10 mM of ATP and MgCl2 was added.

Tag removal
The N-terminal histidine tag was proteolytically removed by incubating approximately 25% of the protein sample (~8 mg/ml in 5 ml) with His-tagged TEV protease at a molar ratio of 50:1 at 20 ºC overnight. The proteolytic reaction went to completion, as judged by SDS-PAGE. Prior to purification, columns were equilibrated with IMAC wash1 buffer (without imidazole) and gel filtration buffer, respectively. Target protein was purified from tag and protease by passing the reaction mixture over a Ni-charged HiTrap Chelating HP column (GE Healthcare). The protein was recovered from flow through and fresh TCEP was added to a final concentration of 2 mM. The protein was subsequently concentrated using an Amicon Ultra-15 centrifugal filter device, 10,000 NMWL (Millipore) and loaded onto the gel filtration column. Fractions containing the target protein were pooled and concentrated to 31.2 mg/ml in a volume of 0.2 ml. The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed and 4000 U Benzonase (Merck), 1 mM ATP and 1 mM MgCl2 was added. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,100 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.2 µl protein solution (diluted to 20 mg/ml) including 2.5 mM ATP and 2.5 mM MgCl2, was mixed with 0.1 µl of well solution consisting of 0.1 M bis-Tris pH 5.5, 0.2 M ammonium acetate and 25% PEG 3350. The plate was incubated at 20 ºC and crystals appeared within 12 days. The crystals were quickly transferred to a cryo solution consisting of well solution complemented with 15% glycerol, and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data was collected at beamline ID 14-2 at the ESRF synchrotron radiation facility in Grenoble, France. Data was indexed and integrated in space group P4322 with the XDS package.
Data Processing:The structure was solved by MR using PDB entry: 2CE7 - metalloprotease FtsH from Thermotoga maritima with Molrep. The asymmetric unit contains one protein monomer. The cell dimensions are a=b=57.20 Å c=155.22 Å. Refmac5 was used for refinement and Coot for model building. TLS restrained refinement using 3 TLS groups was used in the refinement process. The TLS groups were selected using the tlsmd server http://skuld.bmsc.washington.edu/~tlsmd/. Data in the interval 39.13-2.22Å resolution was used and at the end of the refinement the R values were: R= 20.9% and Rfree= 26.2%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 2QZ4.

References

Casari G., De-Fusco M., Ciarmatori S., Zeviani M., Mora M., Fernandez P., DeMichele G., Filla A., Cocozza S., Marconi R., Durr A., Fontaine B. and Ballabio A. (1998). Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93, 973â€“983.
Rugarli E.I. and Langer T. (2006). Translation m-AAA protease function in mitochondria to hereditary spastic paraplegia. Trends Mol. Med. 12, 262-269.
3. Bieniossek C., Schalch T., Bumann M., Meister M., Meier R. and Baumann U. (2006). The molecular architecture of the metalloprotease FtsH. Proc. Nat. Acad. Sci. 103, 3066-3071.