Human Intersectin 2, RhoGEF domain

Target ID:

ITSN2

Deposition Date:

Thursday 26th February 2009

Families:

G-Protein Regulators

Related Diseases:

NeurobiologySignalling

Structure type:

apo

Download Coordinates:

3GF9

Authors:

Shen Y, Tong Y, Tempel W, Li Y, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park HW

Site:

Toronto

ICM Datapack:

ICM Datapack

3GF9

tabs

Structure Details

Intersectin 2, ITSN2, is a 180 kDa scaffold protein containing two EH domains, five SH3 domains, one RhoGEF(Dbl), one PH, and one C2 domain. ITSN2 is involved in clathrin-mediated endocytosis (Pucharcos et al., 2000) and is thought to regulate the formation of clathrin-coated vesicles and may also function in the induction of T-cell antigen recptor endocytosis (McGavin et al., 2001). It was found the K15 protein of human herpesvirus 8 can associate with ITSN2 and colocalize to discrete compartments with the B cell (Lim et al., 2007).

We solved the crystal structure of the RhoGEF domain of ITSN2 using in situ papain proteolysis method. Papain cleaved between the 5th SH3 domain and the RhoGEF domain and only the RhoGEF domain was crystallized.

The structure of the RhoGEF domain of ITSN2 is composed of 8 alpha helices that are folded into an elongated alpha-helix bundle.

Materials and Methods

ITSN2

PDB:3GF9

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC146779
Entry Clone Source:OpenBiosystems EHS1001-99608273
SGC Clone Accession:HPC098-A03
ITSN2:C1103-E1379
Tag:N-terminal tag: mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-V2R pRARE2

Construct

Prelude:Tag not removed
Sequence:mhhhhhhssgrenlyfqgCQVIAMYDYAANNEDELSFSKGQLINVMNKDDPDWWQGEINGVTGLFPSNYVKMTTDSDPSQQWCADLQTLDTMQPIERKRQGYIHELIQTEERYMADLQLVVEVFQKRMAESGFLTEGEMALIFVNWKELIMSNTKLLKALRVRKKTGGEKMPVQMIGDILAAELSHMQAYIRFCSCQLNGAALLQQKTDEDTDFKEFLKKLASDPRCKGMPLSSFLLKPMQRITRYPLLIRSILENTPESHADHSSLKLALERAEELCSQVNEGVREKENSDRLE
Vector:pET28-mhl (GI:134105571)

Growth

Medium:Terrific Broth
Antibiotics:Kanamycin 50 µg/mL + Chloramphenicol 25 µg/mL
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 degC. When OD600 reached ~3.0, the temperature of the medium was lowered to 15 degC 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 degC.

Purification

Procedure
The lysate was centrifuged at 16,000 rpm for 60 minutes and the supernatant was mixed with 6 mL 50% Ni-NTA beads, and incubated at 4 degC on roller drum 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 50 mL binding buffer followed by 50mL washing buffer. The protein bound to beads were then eluted using 15 mL elution buffer. The flow-through was collected and loaded onto Supderdex-75 26/60 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 95% judged by SDS-PAGE.

Extraction

Procedure
Frozen cells from 6L culture were thawed and resuspended in 400 mL Binding buffer with freshly added final concentration of 1mM PMSF/Benzamidine, 0.5% CHAPS and 5U/mL Benzonase (Sigma Catalog # E1014, 250U/&microL), and supplemented with 1mL protease inhibitor cocktail (SIGMA Catalog # P8849), and lysed using sonication at 10 seconds 50% duty cycle for 6 minutes at 120W.
Concentration:31.1 mg/mL
Ligand
MassSpec:Native: 33976.13, expected 33975.77
Crystallization:Stock protein solution was mixed with 1:100 (m/m) papain and then crystallization was setup in sitting drops with Red Wings and SGC-I screens kits. Rod-shaped crystals were seen at multiple PEG conditions, includes RW-A2 and SGC-G9. Crystal used for data collection was grown in 25% PEG 3350, 0.1M (NH4)2SO4, 0.1M Bis-Tris pH 5.5. Sitting drop setup. 0.3 µL protein (include 1:100 papain) + 0.3 uL well solution equilibrated with 100 µL buffer.

SDS-PAGE gel shows the protein in the crystal has a size of 23 kDa, which is about 10 kDa smaller than expected. The original construct include an SH3 and an GEF domain, only the GEF domain is crystallized.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

LIM, C. S., SEET, B. T., INGHAM, R. J., GISH, G., MATSKOVA, L., WINBERG, G., ERNBERG, I., & PAWSON, T. (2007). The K15 protein of Kaposi's sarcoma-associated herpesvirus recruits the endocytic regulator intersectin 2 through a selective SH3 domain interaction. Biochemistry, 46(35), 9874-9885.
MCGAVIN, M. K., BADOUR, K., HARDY, L. A., KUBISESKI, T. J., ZHANG, J., & SIMINOVITCH, K. A. (2001). The intersectin 2 adaptor links Wiskott Aldrich Syndrome protein (WASp)-mediated actin polymerization to T cell antigen receptor endocytosis. J.Exp.Med., 194(12), 1777-1787.
PUCHARCOS, C., ESTIVILL, X., & DE LA, L. S. (2000). Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis. FEBS Lett., 478(1-2), 43-51.