Human ADP-ribosylation factor GTPase activating protein 1

Target ID:

ARFGAP1

Deposition Date:

Tuesday 22nd July 2008

Families:

G-Protein Regulators

Related Diseases:

CancerNeurobiology

Structure type:

apo

Download Coordinates:

3DWD

Authors:

Nedyalkova L, Tong Y, Tempel W, Landry R, Arrowsmith CH, Edwards AM, Bountra C, Wilkstrom M, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3DWD

tabs

Structure Details

Human ARFGAP1 consists of a 406 a.a. with the N-terminal region about 130 a.a. ArfGAP domain that stimulates the GTPase activity of ARF proteins and the C-terminal glycine and serine rich region which is predicted to be unstructured. The ARFGAP1 protein associates with the Golgi apparatus and plays a central role in the formation of coat protein (COPI) vesicles from Golgi membrane[1].

We solved the crystal structure of the N-terminal ArfGAP domain of ARFGAP1. The structure resembles previous structures of ArfGAP domains. A C4 type Zn-finger comprised of Cys22, 25, 42, 45 provides stability to the whole structure.

Materials and Methods

ARFGAP1

PDB:3DWD

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC028233
Entry Clone Source:MGC AT50-C10
SGC Clone Accession:HPC039-A02
Tag:mgsshhhhhhssglvpr*gs, Thrombin cleavage tag
Host:BL21-CodonPlus(DE3)-V2R

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMASPRTRKVLKEVRVQDENNVCFECGAFNPQWVSVTYGIWICLECSGRHRGLGVHLSFVRSVTMDKWKDIELEKMKAGGNAKFREFLESQEDYDPCWSLQEKYNSRAAALFRDKVVALAEGREWSLES
Vector:pET28a-LIC (GI:145307000)

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 60 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 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 15 mL washing buffers(contains 5mM, 30mM or 75 mM Imidazole separately). The protein bound to beads were eluted using 15 mL elution buffer once. The flow-through fractions washed using buffer containing 30mM, 75mM, 300mM Imidazole were collected and loaded onto Supderdex-75 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 85% judged by SDS-PAGE. High molecular weight bands were seen at 56 kDa-130kDa that cannot be separated using gel filtration.

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 microfluidizer.
Concentration:66.6 mg/mL
Ligand
ZnMassSpec:Native: 16691.20, expected 16822.02, the N-terminal Met was removed when expressed in E.coli.
Crystallization:Crystallization was setup using sitting drops with Red Wings and SGC-I screens and also with in situ proteolytic treatment. The crystal used for data collection was grown in 3.5M Sodium Formate 0.1M Tris pH 8.5 and in the presence of 1:100 Chymotrypsin. The crystals grow to a mountable size within one week and shows 2D-plate shape. 2.0 M Li2SO4 or 4.2M NaCl were used as cryoprotectant.
last updated by ytong 20080729
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Lee S.Y., Yang J.S., Hong W., Premont R.T., and Hsu V.W. (2005). ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation. J. Cell Biol. 168: 281-290.