Human chimerin 1

Target ID:

CHN1

Aliases:

ARHGAP2CHNRHOGAP2

Deposition Date:

Thursday 24th April 2008

Families:

G-Protein Regulators

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

3CXL

Authors:

Shen L, Buck M, Tong Y, Tempel W, MacKenzie F, Arrowsmith CH, Edwards AM, Bountra C, Wilkstrom M, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3CXL

tabs

Structure Details

Chimerin 1, (CHN1), also known as alpha-1-chimerin, n-chimerin, or ARHGAP2, is a brain-specific GTPase-activating protein (GAP)[1]. CHN1 is a three-domain protein with the N-terminal SH2 domain, the C-terminal RhoGAP domain and the central C1 domain similar to protein kinase C. When lipid diacylglycerol (DAG) binds to the C1 domain, CHN1 is transferred to the plasma membrane and negatively regulates Rho-family small GTPases RAC1 and CDC42, thus causing the morphological change of axons by pruning the ends of axon dendrites[2,3]. Mutational analysis suggests that un-overlapping residues of the RhoGAP domain are 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 therapeutics[5].

We solved the crystal structure of the full length CHN1. CHN1 adopts an L-shaped configuration of the three domains. The C1 domain contains two Zn2+-fingers. Each Zn2+ ion is coordinated by the side chains of three cysteines and one histidine (H206, C236, C239, and C255 for the first Zn2+ and H244, C219, C222, and C247 for the second Zn2+). An N-terminal helix of the SH2 domain is folded back into the cleft between the RhoGAP domain and the C1 domain and may presents a regulation mechanism for the cross-talking between the three domains.

Materials and Methods

CHN1

PDB:3CXL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC011393
Entry Clone Source:MGC AT8-G1
SGC Clone Accession:HPC074-A05
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-RIL

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgVWKSYLYQLQQEAPHPRRITCTCEVENRPKYYGREFHGMISREAADQLLIVAEGSYLIRESQRQPGTYTLALRFGSQTRNFRLYYDGKHFVGEKRFESIHDLVTDGLITLYIETKAAEYIAKMTINPIYEHVGYTTLNREPAYKKHMPVLKETHDERDSTGQDGVSEKRLTSLVRRATLKENEQIPKYEKIHNFKVHTFRGPHWCEYCANFMWGLIAQGVKCADCGLNVHKQCSKMVPNDCKPDLKHVKKVYSCDLTTLVKAHTTKRPMVVDMCIREIESRGLNSEGLYRVSGFSDLIEDVKMAFDRDGEKADISVNMYEDINIITGALKLYFRDLPIPLITYDAYPKFIESAKIMDPDEQLETLHEALKLLPPAHCETLRYLMAHLKRVTLHEKENLMNAENLGIVFGPTLMRSPELDAMAALNDIRYQRLVVELLIKNEDILF
Vector:pET28-mhl (GI:134105571)

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and 25 µg/mL chloramphenicol at 37 °reeC. When OD600 reached ~3.0, the temperature of the medium was lowered to 15 µC and the culture was induced with 0.5 mM IPTG. The cells were allowed to grow overnight before they were harvested and flash frozen in liquid nitrogen and stored at -80 °C.

Purification

Procedure
The lysate was centrigued at 15,000 rpm for 45 minutes and the supernatants were mixed with 3 mL 50% Ni-NTA beads, and incubated at 4 &degreeC for 1 hours. The supernatant was then passed through a gravity column (Poly-Prep, Bio-Rad, Catalog #731-1550) and the beads were washed using 10 mL washing buffer twice. The protein bound to beads were eluted using 8 mL elution buffer twice. The flow-through was collected and loaded onto Supderdex-200 gel filtration column. Eluted fractions were pooled and concentrated using amicon centrifugal filter (m.w. cut-off 10,000 ). The purity of the proteins was higher than 97% judged by SDS-PAGE.

Extraction

Procedure
Frozen cells from 1.8L TB culture were thawed and resuspended in 150 mL extraction buffer with freshly added 0.5% CHAPS and 2mM BME, and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 3 &microL benzonase (Sigma Catalog # E1014, 250U/&microL), and lysed using sonication at 20 seconds 50% duty cycle for 5 minutes at 100W.
Concentration:24.2 mg/mL
Ligand
ZnMassSpec:Native: 53721.10, expected 53714.77
Crystallization:Crystallization was setup using sitting drops with Red Wings and SGC-I screens initially. Bipyramid crystals were seen at condition SC04 around one month after setup.
Optimization was done using hanging drop vaporization, it usually takes 10 days or more for crystals to grown.
Crystal used for data collection was grown at 1.4M (NH4)2SO4, 100mM Glycine pH 9.5, 10 mM MgCl2, 5% MPD.

Last updated by ytong 20080428
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.