Human GTPase activating protein

Target ID:

RACGAP1A

Aliases:

HsCYK-4ID-GAPMgcRacGAP

Deposition Date:

Wednesday 14th February 2007

Families:

GTPases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2OVJ

Authors:

L.SHRESTHA,E.PAPAGRIGORIOU,M.SOUNDARARAJAN,J.ELKINS,C.JOHANSSON,F.VON DELFT,A.C.W.PIKE,N.BURGESS,A.TURNBULL,J.DEBRECZENI,F.GORREC,C.UMEANO,A.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,D.A.DOYLE,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2OVJ

tabs

Structure Details

The family of small GTPases are a group of enzymes that do not generate a product in the classical enzyme sense that the product is used in a down stream metabolic pathway. This is reflected in the fact that the enzyme turnover rates for these proteins is relatively low (Ras GTP hydrolytic rate constant for is ~0.0005 per second). These enzymes are actually critical components within numerous cellular signalling pathways and as such the GTP hydrolysis process is harnesses as a molecular switch either activiating or inhibiting downstream signalling pathways depending on the state of the protein; in general the GDP bound form is inactive while the GTP bound form is active.

Finer control of this process is achieved through interactions of the small GTPase with a number of disparate proteins that alter various stages of the GTP hydrolysis cycle. Two major families of proteins that bind and regulate GTPase activity are the G-protein Activating Proteins (GAPs) and the Guanine nucleotide Exchange Factors (GEFs). GAPs bind to the small GTPase in the active GTP bound state and enhance the catalytic process whiles GEFs bind in the post GTP hydrolytic state and aid the replacement of GDP for GTP thus converting the enzyme back into the activated state.

RACGAP1 (alias MgcRacGAP) is a GAP protein for the Rho subfamily of GTPases in particular RAC1, RAC2 and CDC42 (Toure et al, 1998). This specificity can be modified by phosphorylation of Ser387 by the Aurora B kinase converting RACGAP1 into a RhoGAP capable of enhancing RhoA GTPase activity. (Minoshima et al, 2003). Functionally, RACGAP1 is a central component in the process of cytokinesis (Niiya et al, 2005; Zhao & Fang, 2005).

Materials and Methods

RACGAP1

PDB:2OVJ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:RACGAP1A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal, TEV cleavable hexahistidine tag
Host:BL-21 (DE3)R3 Rosetta strain

Construct

Prelude:
Sequence:smEGMLADFVSQTSPMIPSIVVHCVNEIE QRGLTETGLYRISGCDRTVKELKEKFLRV KTVPLLSKVDDIHAICSLLKDFLRNLKEP LLTFRLNRAFMEAAEITDEDNSIAAMYQA VGELPQANRDTLAFLMIHLQRVAQSPHTK MDVANLAKVFGPTIVAHAVPNPDPVTMLQ DIKRQPKVVERLLSLPLEYWSQFMMVE
Vector:

Growth

Medium:
Antibiotics:
Procedure:The RACGAP1A-c011 glycerol stock was used to innoclate 20 ml LB media with 50 microG/ml kanamycin and 34 microG/ml chloramphenicol which was placed in a 37degC shaker overnight. The next day this starter culture was used to innoculate 1 litre of TB medium which contained 50 µg/ml kanamycin. When OD600 reached ~1.1 the temperature was shifted down from 37degC to 22degC for 1 hour before induction with the addition of 0.5 mM IPTG. Protein expression was allowed to carry on for a further 16 hours before harvest.

Purification

Procedure
Column 1: IMAC Ni-NTA column purification
Procedure: The Supernatant containing the protein was loaded on to manually packed Ni sepharose beads column and washed with 30 ml of wash buffer and eluted in 6 fractions of 2 ml each using the high imidazole elute buffer. The eluate was pooled and concentrated using 10 KDa cutoff millipore filters and loaded on to Gelfiltration column

Column 2 : Gel filtration, Hiload 16/60, S75 16/60 - 120 ml
Procedure: The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.0 ml/min. Eluted proteins were collected in 1 ml fractions. The fractions containing protein were identified on a coomasie blue stained gel.

Enzymatic treatment : Add 100 µl of the home produced TEV protease to fractions containing protein and left at 4°C overnight. The protein was rebound to Nickel beads for removing the His tag and the flow through containing RACGAP1A

Concentration : The concentration of RACGAP1A was determined to be 25 mg/ml

Extraction

Procedure
Total vol of cell: 60 mls (estimate). Cell breakage: After thawing, the resuspended cells were lysed by passing through Emulsiflex C5 high pressure homogeniser. PEI (stock 5 %) was added to the homogenate to a final concentration of 0.15%. The cell debris, nuclei and DNA were spun down at 16000 rpm for 45 min. The supernatant was collected. Discard pellet.
Concentration:
Ligand
MassSpec:The Mass of the purified protein was measured to be 22719.4 which matched with the expected mass of 22720.3
Crystallization:Crystals grew from 2:1 ratio mix of protein and precipitant containing 25 % PEG3350, 0.1M Bis Tris 5.5.
NMR Spectroscopy:
Data Collection:Resolution: 1.7 Å, X-ray source: Synchrotron SLS-X10, single wavelength
Data Processing:

References

Toure A, Dorseuil O, Morin L, Timmons P, Jegou B, Reibel L, Gacon G. (1998). MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells. J. Biol. Chem. 273: 6019-6023.
Minoshima Y, Kawashima T, Hirose K, Tonozuka Y, Kawajiri A, Bao YC, Deng X, Tatsuka M, Narumiya S, May WS Jr, Nosaka T, Semba K, Inoue T, Satoh T, Inagaki M, Kitamura T. (2003). Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis. Dev. Cell. 4: 549-560.
Niiya F, Xie X, Lee KS, Inoue H, Miki T. (2005). Inhibition of cyclin-dependent kinase 1 induces cytokinesis without chromosome segregation in an ECT2 and MgcRacGAP-dependent manner. J. Biol. Chem. 280 : 36502-36509.
Zhao WM, Fang G. (2005). MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis. Proc. Natl. Acad. Sci. U S A. 102: 13158-13163.