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Human GRP1 (general receptor for phosphoinositides 1)-associated scaffold protein

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PDB Code 2PNT Target Class Linkers

Target GRASPA
Alias TAMALIN
Disease Area/Function signalling, neurobiology
Date Deposited 2007-04-25
Authors J.ELKINS, E.PAPAGRIGORIOU, C.COOPER, C.GILEADI, J.UPPENBERG, J.BRAY, F.VON DELFT, A.C.W.PIKE, E.UGOCHUKWU, C.UMEANO, O.GILEADI, A.EDWARDS, C.H.ARROWSMITH, J.WEIGELT, M.SUNDSTROM, D.A.DOYLE, STRUCTURAL GENOMICS CONSORTIUM (SGC)

Struc Details Tabs

Structure Details
PDZ domains function as protein-protein interaction modules. The best characterised interaction of the PDZ domain is with the C-terminal 4-5 residues of the target protein that binds in an extended fashion between the betaB strand and the alphaB helix of the PDZ domain. In many cases the protein that contains the PDZ domain contains additional PDZ domains or interaction modules. GRASP falls into this category as it contains in addition to the PDZ domain a leucine-zipper region and a C-terminal PDZ recognition motif comprising the amino acids ESQL-COO-. As such the rat version of GRASP which is known as tamalin which is expressed in neurons has been shown to interact with a number of neuronal protein including Disks large-associated protein 1 (DAP-1) and DAP-3 (Kitano et al, 2003), the gamma-aminobutyric acid B receptor 2 (Kitano et al, 2002) as well as the metabotropic glutamate receptor (mGluR) 1a and 5 (Kitano et al, 2002).
The crystal structure of tamalin forms a novel dimerisation interaction and was determined in the apo form as well as in the presence of the C-terminal peptide from mGluR5 (Sugi et al, 2007). Here we present the crystal structure of human GRASP in complex with the C-terminal peptide of mGluR1. This crystal structure is also a dimer however the dimerisation interface is different from that observed by Sugi and colleagues and the mode of peptide binding for the "unconventional binding mode" differs markedly.

References

  1. Kitano J, Kimura K, Yamazaki Y, Soda T, Shigemoto R, Nakajima Y, Nakanishi S. (2002). Tamalin, a PDZ domain-containing protein, links a protein complex formation of group 1 metabotropic glutamate receptors and the guanine nucleotide exchange factor cytohesins. J. Neurosci. 22: 1280-1289.
  2. Kitano J, Yamazaki Y, Kimura K, Masukado T, Nakajima Y, Nakanishi S. (2003). Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association. J. Biol. Chem. 278: 14762-14768.
  3. Takuma Sugi, Takuji Oyama, Takanori Muto, Shigetada Nakanishi, Kosuke Morikawa and Hisato Jingami (2007). Crystal structures of autoinhibitory PDZ domain of Tamalin: implications for metabotropic glutamate receptor trafficking regulation The EMBO Journal 26: 2192-2205.
Materials & Methods
StructureGRASP
PDB Code2PNT
Entry clone accession gi|32171221
Entry clone source MGC
SGC clone accession GRASPA-c008
Tag N-terminal, TEV cleavable hexahistidine tag
Construct sequencesmQQRKVLTLEKEDNQTFGFEIQTYGLHHREEQRVEMVTFVCRVHESSPAQLAGLTPGDTIASVNGLNVEGIRHREIVDI
IKASGNVLRLETLYSSTL
Vector pNIC28-Bsa4.
Expression host BL21(DE3)-R3-pRARE2 (A homemade phage resistant version of BL21(DE3) containing the pRARE2 plasmid from Rosetta II (DE3) cells).
Growth method The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure. A number of colonies from the transformation were used to innoculate 1 mL of LB media containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.
A glycerol stock was used to innoculate 40 mL of LB media containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to innoculate 2x 1L of TB media (18 mL starter culture into each) containing 50 µg/mL kanamycin. After 5 hours the temperature was reduced to 18°C. After a further 1.5 hours the cells were induced by the addition of 1.0 mM IPTG. The expression was continued overnight.
Cells were spun at 6000 rpm, JLA8.1000 rotor, for 15 mins at 4°C. The cell pellets were placed in a -80°C freezer
Extraction buffersLysis Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole pH 7.4, 0.5 mM TCEP
Extraction procedure The cell pellet was resuspended in 50 mL of lysis buffer containing 0.5 mM PMSF. The resuspended cell pellet was passed 4 times through an Emulsiflex C5 high-pressure homogeniser, collecting a final volume of approximately 200 mL after dilution with Lysis Buffer. PEI was added to a final concentration of 0.2 % and the cell debris and precipitated DNA were spun down (17500 rpm, JA18 rotor, 90 min). The supernatent was filtered through a 0.2 m M syringe filter.
Purification buffersBinding Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole pH 7.4, 0.5 mM TCEP;
Wash Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 25 mM Imidazole pH 7.4, 0.5 mM TCEP;
Elution Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4, 0.5 mM TCEP.
Gel Filtration buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 0.5 mM TCEP
Purification procedure Column 1: HisTrap 1mL.
Column 2: Gel filtration. Hiload S200 16/60 - 120 mL volume.
The protein was purified using an AktaExpress system.
The clarified cell extract was passed through the column 1 at a flow rate of 0.8 mL/min. The column was then washed Binding Buffer until a stable UV baseline was achieved. The column was then washed with Wash Buffer until a stable UV baseline was achieved. The protein was eluted with 5 mL of Elution Buffer.
The column 2 was pre-equilibrated with Gel Filtration Buffer. The HisTrap eluant was loaded on the gel filtration column automatically after the HisTrap elution at a flow rate of 1.2 mL/min. Eluted proteins were collected in 1.8 mL fractions. The fractions containing protein were identified on a coomasie blue stained gel.
TEV protease digestion: The gel filtration fractions containing GRASPA were pooled and 170 µl of TEV protease solution (about 1 mg/mL) was added. The digestion was left overnight at 4°C.Rebinding of impurities to Ni-NTA: The protein was mixed with Ni-NTA resin (0.25 mL, pre-equilibrated into Gel Filtration Buffer) at 4°C for 60 minutes. The resin was spun down and the supernatent collected.
Protein stock concentration The TEV protease cleaved GRASPA was concentrated to 50 mg/ml (measured using a nanodrop machine), distributed into aliquots and frozen at -80°C.
Mass spec Measured: 11038.3; Expected: 11038.4
Crystallization Crystals grew from a 2:1 ratio of protein to precipitant solution (1.26 M NaH2PO 4 , 0.14 M K 2 HPO 4 ), using the vapour diffusion method.
Data collection Crystals were cryo-protected by equilibration into precipitant solution containing 30% glycerol, and then flash frozen in liquid nitrogen. Data was collected to a resolution of 2.15 Å at the Swiss Light Source, beamline X10SA.
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