Human membrane associated guanylate kinase, WW and PDZ domain containing 1 (6th domain)

Target ID:

MAGI1A@6

Aliases:

AIP3BAIAP1BAP1MAGI-1TNRC19WWP3

Deposition Date:

Wednesday 19th December 2007

Families:

Linkers

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

3BPU

Authors:

Pilka, E.S., Hozjan, V., Cooper, C., Pike, A.C.W., Elkins, J., Doyle, D.A., von Delft, F., Arrowsmith, C.H., Weigelt, J., Edwards, A., Oppermann, U.

Site:

Oxford

ICM Datapack:

ICM Datapack

3BPU

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 also possesses additional PDZ domains or interaction modules. MAGI1 is one such protein that contains a guanylate kinase domain, WW domains and five PDZ domains within its sequence (Dobrosotskaya et al, 1997).

MAGI1 is one of the components of the closely associated areas between cells that form tight junctions (Dobrosotskaya and James, 2000). It along with the small GTPase Rap1 plays a critical role in adherens junction maturation (Sakurai et al, 2006) and development of the central nervous system through delta-notch signaling at adherens junctions (Mizuhara et al, 2005). It also has an important role to play in apoptosis which involves the break down of cell-cell tight juctions by the cleavage of MAGI1 by the proteases caspases-3 and -7 (Gregorc et al, 2007).

Here we present the crystal structure of the sixth PDZ domain of MAGI1 bound to a C-terminal peptide derived from beta-catenin (Dobrosotskaya and James, 2000). Based on homology modelling it was noted that the side chain for Cys1190 in the 6th PDZ domain of MAGI1 was likely to be exposed to solvent. In order to prevent poor biochemical behaviour this side chain was mutated into a Ser residue.

Materials and Methods

MAGI1

PDB:3BPU

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:n/a
Entry Clone Source:Synthetic
SGC Clone Accession:
Tag:N-terminal TEV-cleavable (at *) his-tag with the following sequence mhhhhhhssgvdlgtenlyfq*s
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence: sMELITVHIVKGPMGFGFTIADSPGGGGQRVKQIVDSPRSRGLKEGDLIVEVNKKNVQALTHNQVVDMLVESPKGSEVTLLVQRQTRL
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:An overnight culture (10 ml) was used to innoculate 1L TB medium (supplemented with 50 µg/ml of Kanamycin). The cells were cultured at 37°C with vigorous shaking (160 rpm) until the culture reached an OD600 of 1.5. At that point temperature was reduced to 18°C, and cells were induced with IPTG at a concentration of 0.5 mM, and cultured further for 18 hours. Cells were harvested at 6000 rpm for 10 minutes and the cell pellet of 1L was resuspended in 20 ml of lysis buffer and stored at -20°C until further use. Lysis buffer : 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 5 mM imidazole, Complete TM EDTA-free protease inhibitor (Roche, 1tablet / 50ml).

Purification

Procedure

Column 1: Ni-NTA

Buffers: Binding buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5,

5 mM imidazole, 0.5mM TECP; Wash Buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 30 mM imidazole, 0.5mM TECP; Elution Buffer: 500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 250 mM imidazole, 0.5mM TECP.

Procedure: A 0.5ml NiNTA column was equilibrated with 12 ml of Binding buffer. The lysed sample was applied to the column twice and washed through with 12 ml of Binding Buffer (Wash 1) and 25ml of Wash Buffer (Wash 2). The protein was eluted with 15mls of Elution buffer

Column 2: Size Exclusion Chromatography (SEC) Hiload 16/60 Superdex 200 prep grade 120 ml (GE/Amersham Biosciences)

Buffers: Gelfiltration bufffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5mM TCEP.

Procedure: AKTA Purifier Gel Filtration. The eluted fractions from the Ni-NTA column were loaded on the gel filtration column in Gelfiltration buffer at 1ml/min. The flow rate was 1ml/min and the pure protein was eluted at 65-82min. The protein was collected in a 96 well block and analyzed by SDS-PAGE. Positive fractions were pooled for TEV cleavage.

TEV cleavage: The gel filtration fractions containing MAGI1A were pooled and 150µl of TEV protease solution (9mg/ml) was added. The digestion was left overnight at 4°C and cleavage was examined by SDS page, before passing the mixture through Ni-NTA resin.

Extraction

Procedure
The resuspended pellet was thawed and lysed in a high pressure homogeniser and then centrifuged at 4°C for 45 minutes at 4.8g.
Concentration:The combined samples from the SEC column (identified by SDS PAGE) were concentrated using centricons with 5 kDa cut off (Amicon Ultra 5k, Millipore) to 2 mg/ml.
Ligand
MassSpec:LC-ESI-MS tof confirmed the correct mass of 9537 Da expected for this construct of MAGI1A
Crystallization:Crystals were grown by vapor diffusion at 20°C in 300nl sitting drops. The drops were prepared by mixing 200nl of protein solution and 100nl of precipitant consisting of 0.05M Zn acetate pH 6.2 and 30% PEG 3350. Crystals were transferred to a cryo-protectant consisting of the well solution and 25% glycerol before flash-cooling in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 1.6Å; X-ray source: SLS beam X10SA
Data Processing:

References

Dobrosotskaya I, Guy RK, James GL. (1997). MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interaction domains. J. Biol. Chem. 272: 31589-31597.

Dobrosotskaya IY, James GL. (2000). MAGI-1 interacts with beta-catenin and is associated with cell-cell adhesion structures. Biochem. Biophys. Res. Commun. 270: 903-909.

Gregorc U, Ivanova S, Thomas M, Guccione E, Glaunsinger B, Javier R, Turk V, Banks L, Turk B. (2007). Cleavage of MAGI-1, a tight junction PDZ protein, by caspases is an important step for cell-cell detachment in apoptosis. Apoptosis. 12: 343-354.

Mizuhara E, Nakatani T, Minaki Y, Sakamoto Y, Ono Y, Takai Y. (2005). MAGI1 recruits Dll1 to cadherin-based adherens junctions and stabilizes it on the cell surface. J. Biol. Chem. 280: 26499-26507.

Sakurai A, Fukuhara S, Yamagishi A, Sako K, Kamioka Y, Masuda M, Nakaoka Y, Mochizuki N. (2006). MAGI-1 is required for Rap1 activation upon cell-cell contact and for enhancement of vascular endothelial cadherin-mediated cell adhesion. Mol. Biol. Cell. 17: 966-976.