Human Rho GTPase Activating Protein 15

Target ID:

ARHGAP15A

Aliases:

ARHGAP15BM046

Deposition Date:

Wednesday 16th January 2008

Families:

GTPases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

3BYI

Authors:

Shrestha, L., Tickle, J., Elkins, J., Burgess-Brown N., Johansson, C., Papagrigoriou, E., Kavanagh, K., Pike, A.C.W., Ugochukwu, E., Uppenberg, J., von Delft, F., Arrowsmith, C.H., Edwards, A., Weigelt, J. Doyle, D.

Site:

Oxford

3BYI

tabs

Structure Details

GTPases are essential components within numerous cellular pathways and the Rho members of this family are responsible in the control of signaling pathways linking membrane receptors to the assembly and disassembly of adhesion complexes and actin cytoskeletal dynamics and gene regulation through down stream effectors. Owing to its wide range of cellular functions, fine regulation of the signaling process is highly necessary, which is achieved through a specific group of proteins termed RhoGAPs or ARHGAPs. GTPase activating proteins (GAPs) are encoded by approximately 0.5 % of the total genes encoded in human and function through directly interacting with GTP bound active GTPase in the transition state and accelerate the hydrolysis up to 105 times. ARHGAP15 have been reported to function as a GAP for Rac1 GTPase but the physiological role of the GAP still remains unknown.

We have determined the structure of ARHGAP15, which is completely alpha helical. Its structure is similar to other RhoGAP members containing four bundled helices, with a conserved arginine residue that participates directly in catalysis by providing the ‘arginine finger’ residue. This residue protrudes into the GTPase active site and positions the catalytically vital Gln 61 residue in the switch II region during transition state, accelerating the catalytic cycle.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:5553184

SGC Construct ID: ARHGAP15A-c002

GenBank GI number: gi|28466979

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq*sMRPSLK
TLQEKGLIKDQIFGSHLHKVCERENSTVP
WFVKQCIEAVEKRGLDVDGIYRVSGNLAT
IQKLRFIVNQEEKLNLDDSQWEDIHVVTG
ALKMFFRELPEPLFPYSFFEQFVEAIKKQ
DNNTRIEAVKSLVQKLPPPNRDTMKVLFG
HLTKIVAKASKNLMSTQSLGIVFGPTLLR
AENETGNMAIHMVYQNQIAELMLSEYSKI
FGSE

Host:BL21(DE3)-R3-pRARE2 (previously known as Rosetta)

Growth medium, induction protocol: 10ml from a 50 ml overnight culture containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol was used to inoculate 1 liter of TB media containing the same concentration of antibiotics. Cultures were grown at 37°C until the OD₆₀₀ reached ~1.5. After that the temperature was adjusted to 22°C. Expression was induced over night using 0.5 mM IPTG. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5% glycerol, 5 mM imidazole.

Extraction method: Cell pellets were lysed by C5 high pressure homogenizer (Avestin). The lysate was centrifuged at 17500 rpm for 60 minutes and the supernatant collected for purification.

Column 1: Ni-affinity chromatography.

Buffers: Binding buffer: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 5mM Imidazole. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% glycerol. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, 5% Glycerol.

Procedure: 5 ml of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 50 ml binding buffer. The lysate was applied to the column which was subsequently washed with 50 ml wash buffer 1 and 2. The protein was eluted by gravity flow by applying 15 ml portions of elution buffer containing 250 mM imidazole. The eluted protein was analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 5mM.

Column 2: Size exclusion chromatography (Superdex S200, 60 x 1cm)

SEC-Buffers: 50 mM Hepes, pH 7.4, 500 mM NaCl, 0.5 mM TCEP.

Procedure: The fractions eluted of the Ni-affinity chromatography were concentrated to about 2 mls using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S200 column equilibrated in SEC buffer at a flow rate of 0.8 ml/min. Eluted fractions were >95% pure as judged by SDS-PAGE.

Protein concentration: Centricon with a 10 kDa cut off in SEC-buffer.

Mass Spectrometry Characterisation: The mass of the protein was calculated to be 24472.1 Da and experimentally determined mass was 24472.5 Da for the His tag cleaved protein. The identity of the protein was reconfirmed to be correct by DNA sequencing both DNA strands of this expression construct.

Crystallization: The protein was concentrated in gel filtration buffer to a protein concentration of 17.48 mg/ml. Crystallizations were carried out using sitting drop vapor diffusion at 20°C. Crystals appeared in 150nl drops composed of equal volume of protein (17.5mg/ml) and reservoir solution containing 2.4 M sodium malonate 7.0.

Data Collection: Crystals were cryo-protected using the well solution supplemented with 20% Ethylene Glycol and flash frozen in liquid nitrogen.Resolution: 2.2 Å; Diffraction data were collected at the SLS beam line SAX10.

References

Hall A. (2005) Rho GTPases and the control of Cell behaviour. Biochem Soc Trans. 33: 891-895.

Rittinger K, Walker PA, Eccleston JF, Smerdon SJ, Gamblin SJ. (1997) Structure of
1.65 A of RhoA and its GTPase-activating protein in complex with transition-state. Nature 389(6652): 758-762.

Seoh ML, Ng CH, Yong J, Lim L, Leung Y. (2003) ArhGAP15, a novel human RacGAP protein with GTPase binding property. FEBS Lett 539(1-3):131-137.