Human phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1, first PDZ domain

Target ID:

PREX1

Deposition Date:

Thursday 27th January 2011

Families:

G-Protein Regulators

Related Diseases:

CancerMetabolism

Structure type:

apo

Download Coordinates:

3QIK

Authors:

Shen L, Tong Y, Tempel W, Li Y, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Park H

Site:

Toronto

3QIK

tabs

Structure Details

PREX1 is a multi-domain cytoplasmic guanine nucleotide exchange factor for the Rho family of small GTPases. It can be activated by phosphatidylinositol-3,4,5-trisphosphate or the β, γ subunits of heteromeric G proteins [1]. PREX1 is highly overexpressed in human breast cancer cells and mediates ErbB-driven tumorigenesis [2]. PREX1 is also a candidate gene for type 2 diabetes [3] and links mTOR signaling to Rac activation and cell migration [4]. PREX1 contains two tandem PDZ domains and they interact with the C-terminus of a new family of GPCRs, sphignosine-1-phosphate receptors (S1PRs), and promotes the formation of functional aggregates of S1PRs [5].

The first PDZ domain of PREX1 was expressed in E. coli and purified using IMAC affinity chromatography. The crystal structure of the PDZ domain was solved at 2.3 Å resolution. The PDZ domain forms a dimer through the N-terminal helix. This mode of dimerization is not seen in other known dimeric PDZ domain structures, e.g. the PDZ domains of Tamalin, Zonula occludens, HtrA proteins. This suggests PREX1 PDZ domains may promote the functional aggregation of S1PRs through a novel mechanism.

Materials and Methods

PREX1

PDB:3QIK

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC053616
Entry Clone Source:Open Biosystems
(actual DNA used HPC0A0-B07, a chimeric full length DNA of BC053616 and synthesized DNA)
SGC Clone Accession:HPC0AJ-H05
Tag:N-terminal His6-tag, removed before crystallization
Host:BL21-V2R-pRARE2

Construct

Prelude:PREX1:K607-A706
Sequence:gKNKQLRNDFKLVENILAKRLLILPQEEDYGFDIEEKNKAVVVKSVQRGSLAEVAGLQVGRKIYSINEDLVFLRPFSEVESILNQSFCSRRPLRLLVATKA
Vector:pET28-MHL

Growth

Medium:Terrific Broth medium in the presence of 50 mg/mL kanamycin and 25 mg/mL chloramphenicol
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 2 L of growth medium at 37 °C. When OD₆₀₀ reached ~3.0, the temperature of the medium was lowered to 15 °C and the culture was induced with 1 mM IPTG. The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen and stored at -80 °C.

Purification

Procedure
The lysate was centrifuged at 15,000 rpm for 45 minutes and the supernatant were mixed with 4 mL 50% flurry of Ni-NTA beads and incubated at 4 degree Celsius on rotary shaker for one hour. The mixture was then centrifuged at 2300 rpm for 5 min and the supernatant discarded. The beads were then washed with 50 mL binding buffer containing 5 mM Imidazole twice. Bound proteins were eluted using 15 mL elution buffer. The flow-through was collected and further purified by a Superdex-75 gel filtration column pre-equilibrated with gel filtration buffer. Fractions containing the target protein were pooled and concentrated using Amicon Ultra-15 centrifugal filter (mwco 10 kDa). The purity of the preparation is tested by SDS-PAGE to be greater than 95%.

Extraction

Procedure
Frozen cells from 4L TB culture were thawed and resuspended in 5 mL extraction buffer per gram of cell pellets with freshly added 0.5% CHAPS, and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 3 uL benzonase (Sigma Catalog # E1014, 250U/uL), and lysed using sonication for 5 min at 100 W, 10 sec on/10 sec off duty cycle.
Concentration:6.9 mg/mL
Ligand
MassSpec:Uncut version native protein expected 13638.7, measured 13639.0
Cut version, expected 11502.4, measured 11494.5, not sure about the cause of -7.9 difference
Crystallization:Crystal was initially obtained from SGC-I screen condition B12.
Crystal used for structure refinement was grown in 1.2 M Li₂SO₄, 0.5 M (NH₄)₂SO₄, 0.1 M Sodium Citrate pH 6.2 in sitting drop setup, using 1 uL protein + 1 uL well solution against 1 mL reservoir buffer at room temperature. Crystal used for phasing was Pt derivative of optimized crystal grown in 0.5 M (NH₄)₂SO₄, 1.2 M Li₂SO₄, 0.1 M Sodium Citrate pH 5.6. Crystal was harvested from original drop and soaked in 4 uL of the mother liquid containing 1 mM K₂PtI₆ for 6 hours and harvested for diffraction test.

Crystals grow to a mountable size within one week.
Cryo used paratone-N
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Welch HC, Coadwell WJ, Ellson CD, Ferguson GJ, Andrews SR, Erdjument-Bromage H, Tempst P, Hawkins PT, Stephens LR. P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac. Cell 2002, 108:809-821.
Montero JC, Seoane S, Ocana A, Pandiella A. P-Rex1 participates in Neuregulin-ErbB signal transduction and its expression correlates with patient outcome in breast cancer. Oncogene 2011, 30:1059-1071.
Bento JL, Palmer ND, Zhong M, Roh B, Lewis JP, Wing MR, Pandya H, Freedman BI, Langefeld CD, Rich SS, Bowden DW, Mychaleckyj JC. Heterogeneity in gene loci associated with type 2 diabetes on human chromosome 20q13.1. Genomics 2008, 92:226-234.
Hernandez-Negrete I, Carretero-Ortega J, Rosenfeldt H, Hernandez-Garcia R, Calderon-Salinas JV, Reyes-Cruz G, Gutkind JS, Vazquez-Prado J. P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration. J Biol Chem. 2007, 282:23708-23715.
Ledezma-Sanchez BA, Garcia-Regalado A, Guzman-Hernandez ML, Vazquez-Prado J. Sphingosine-1-phosphate receptor S1P1 is regulated by direct interactions with P-Rex1, a Rac guanine nucleotide exchange factor. Biochem. Biophys. Res. Commun. 2010, 391:1647-1652.