Human chimerin 1 Rho-GAP domain

Target ID:

CHN1

Aliases:

ARHGAP2CHNRHOGAP2

Deposition Date:

Monday 5th February 2007

Families:

GTPases

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

2OSA

Authors:

J.R.WALKER,B.S.HONG,L.SHEN,C.H.ARROWSMITH,M.SUNDSTROM,J.WEIGELT,A.M.EDWARDS,A.BOCHKAREV,H.W.PARK,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2OSA

tabs

Structure Details

Chimerin 1, or CHN1, also known as alpha-1-chimerin, n-chimerin or ARHGAP2, is a brain GTPase-activating protein identified by Hall et al. in 1990[1].
CHN1 is a three-domain protein with an N-terminal SH2 domain, a C-terminal RhoGAP domain and a central C1 domain homologous to protein kinase C.
Upon binding with lipid diacylglycerol (DAG) to the C1 domain, CHN1 is relocalized to the plasma membrane and negatively regulates Rho-family small GTPases RAC1 and CDC42, thus causing morphological change of axons by pruning the ends of axon dendrites[2,3].
Mutational analysis suggest different residues of the RhoGAP domain may be involved in RAC1-binding and the RAC1-GAP activity [3,4].
Regulation of the RhoGAP activity of CHN1 by phorbol esters, natural compounds mimic of the lipid second messenger DAG, presents a possible way of designing agents for therapeutic applications[5].

Materials and Methods

CHN1

PDB:2OSA

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_001813
Entry Clone Source:MGC clone AT8-G1
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with thrombin cleavage site: mhhhhhhssglvprgs
Host:E.coli BL21 (DE3) codon plus RIL

Construct

Prelude:
Sequence:gsKHVKKVYSCDLTTLVKAHTTKRPMVVDMCIREIESRGLNSEGLYRVSGFSDLIEDVKMAFDRDGEKADISVNMYEDINIITGALKLYFRDLPIPLITYDAYPKFIESAKIMDPDEQLETLHEALKLLPPAHCETLRYLMAHLKRVTLHEKENLMNAENLGIVFGPTLMRSPELDAMAALNDIRYQRLVVELLIKNEDILF
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:The target was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into 1.8 L of Terrific Broth medium in the presence of 50 microG/mL kanamycin and chloramphenicol at 37degC. When OD600 was ~3.0, the culture was induced with 1mM IPTG and the temperature was reduced to 15degC, and the cells were allowed to grow overnight.

Purification

Procedure
The supernatant was loaded onto 5 mL HiTrap Ni-NTA column (Pharmacia Amersham) equilibrated with binding buffer at 4 ºC using peristaltic pump. The HiTrap Ni-NTA column was washed stepwise with 25 mL of binding buffer, 25 mL of binding buffer with 30 mM imidazole, and 25 mL of binding buffer with 50 mM imidazole. The His-tagged protein was eluted with a linear gradient of imidazole from 50 mM to 500 mM in 50 mL. The fractions corresponding to the correct eluted protein peaks were combined. The protein was further purified on a Superdex 75 (GE Healthcare) gel filtration column, equilibrated with gel filtration buffer. Protein peak fractions were combined, and DTT was added to a final concentration of 10 mM. The protein was concentrated using an Amicon Ultra (m.w cutoff 10 kDa) centrifugal filter and the concentration for protein stock solution was estimated by Bradford to be 60 -110 mg/mL (in different batches).

Extraction

Procedure
Cultures were harvested by centrifugation and the cell pellets were stored at -80 ºC before use. Cells from 1.8L culture were thawed and resuspended in 150 mL of binding buffer with 0.5% CHAPS (Sigma) and 1 mM phenylmethyl sulfonyl fluoride (PMSF) and lysed with microfluidizer. The lysate was centrifuged at 38,400 xg for 45 min and collected the supernatant.
Concentration:79.7 mg/mL
Ligand
MassSpec:Measured 23114.29 D, expected 23113.90 D.
Crystallization:Crystallization trials were set up using the sitting drop vapor diffusion method at room temperature. The protein drop was equilibrated against a reservoir solution (1:1 volume ratio) containing 30.0% Jeffamine ED-2001, 0.1M HEPES pH 7.0. Crystals reached a size of about 50 microns within two to three days.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Hall C., Monfries C., Smith P., Lim H.H., Kozma R., Ahmed S., Vanniasingham V., Leung T., and Lim L. (1990). Novel human brain cDNA encoding a 34,000 Mr protein n-chimaerin, related to both the regulatory domain of protein kinase C and BCR, the product of the breakpoint cluster region gene. J. Mol. Biol. 211: 11-16.
Buttery P., Beg A.A., Chih B., Broder A., Mason C.A., and Scheiffele P. (2006). The diacylglycerol-binding protein alpha1-chimaerin regulates dendritic morphology. Proc. Natl. Acad. Sci. U. S. A 103: 1924-1929.
Kozma R., Ahmed S., Best A., and Lim L. (1996). The GTPase-activating protein n-chimaerin cooperates with Rac1 and Cdc42Hs to induce the formation of lamellipodia and filopodia. Mol. Cell Biol. 16: 5069-5080.
Ahmed S., Lee J., Wen L.P., Zhao Z., Ho J., Best A., Kozma R., and Lim L. (1994). Breakpoint cluster region gene product-related domain of n-chimaerin. Discrimination between Rac-binding and GTPase-activating residues by mutational analysis. J. Biol. Chem. 269: 17642-17648.
Kazanietz M.G. (2005). Targeting protein kinase C and "non-kinase" phorbol ester receptors: emerging concepts and therapeutic implications. Biochim. Biophys. Acta 1754: 296-304.