Human membrane associated guanylate kinase, WW and PDZ domain containing 1(7th domain)

Target ID:

MAGI1A@7

Aliases:

AIP3BAIAP1BAP1MAGI-1TNRC19WWP3

Deposition Date:

Thursday 14th June 2007

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

2Q9V

Authors:

Pilka, E.S., Hozjan, V., Kavanagh, K.L., Papagrigoriou, E., Cooper, C., Elkins, J.M., Doyle, D.A., von Delft, F., Sundstrom, M., Arrowsmith, C.A., Weigelt, J., Edwards, A., Oppermann, U.

Site:

Oxford

ICM Datapack:

ICM Datapack

2Q9V

tabs

Structure Details

In vertebrates the membrane associated guanylate kinase family of tumor suppressors (MAGUK) is localised in the tight junctions in epithelial cells and synaptic junctions in neurons. Tight junctions act as a semi-permeable barrier dividing epithelial cells into apical and basolateral membrane domains. The disruption of this barrier directly contributes to carcinogenesis by deregulating normal proliferation and differentiation programs in epithelial cells (Matter et al., 2003).

The membrane associated guanylate kinase inverted (MAGI-1), localised in tight junctions, is one of the caspase targets during early stages of apoptosis. A 97kDa N-terminal fragment generated by caspase cleavage dissociates from the membrane, thereby disrupting cell-cell contacts (Gregorc et al., 2007). MAGI-1 contains 6 PDZ domains, the structure of the 4th one is presented here. PDZ is an acronym combining the first letters of three proteins â€˜post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1)â€™ which were first discovered to share this domain. A Cys to Ser mutation was introduced to prevent this domain from aggregation during the purification process.

PDZ domains function as protein-protein interaction modules. The best characterised interaction of the PDZ domain is with the C-terminal X-T/S-X-V motif of the target protein that binds in an extended fashion between the β2 strand and the α2 helix of the PDZ domain (Doyle et al., 1996).

Materials and Methods

MAGI1

PDB:2Q9V

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|74272282
Entry Clone Source:Synthetic
SGC Clone Accession:MAGI1A-c036
Tag:N-terminal TEV-cleavable (at *) his-tag with the following sequence mhhhhhhssgvdlgtenlyfq*s.
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*sMEQDIFLWRKETGFGFRILGGNEPGEPIYIGHIVPLGAADTDGRLRSGDELISVDGTPVIGKSHQLVVQLMQQAAKQGHVNLTVRQTRL
Vector:pNIC28-Bsa4.

Growth

Medium:LB
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 in 2 litres 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.

Purification

Procedure
Column 1: Ni-affinity, HisTrap, 1 ml (GE/Amersham Biosciences )
Column 2: Hiload 16/60 Superdex 200 prep grade 120 ml (GE/Amersham Biosciences)

AKTA Xpress Affinity/Gel Filtration. The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280nm was automatically collected.

AKTA Xpress Affinity/Gel Filtration. The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 0.80 ml/min. Eluted protein was collected in 1.8 ml fractions in a 96 well bloc and analyzed by SDS-PAGE. Positive fractions were pooled for TEV cleavage.

TEV cleavage: The gel filtration fractions containing MAGI1A were pooled and 350 µl of TEV protease solution (1mg/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 homogenised by sonication and then centrifuged at 4°C in Beckman JA-25.50 rotor for 60 minutes at 21.000(rpm).
Concentration:The protein was concentrated to 8.1 mg/ml by using an Amicon Ultra 5k concentrator (Millipore).
Ligand
MassSpec:The experimentally determined mass of MAGI1A was 9.866Da, which corresponds to the theoretical mass.
Crystallization:Crystals were grown by vapor diffusion in sitting drops at 4°C. A sitting drop consisting of 50 nl protein (8 mg/ml) and 100 nl well solution was equilibrated against well solution containing 20% PEG 3350, 10% ethylene glycol, 200 mM sodium acetate, 100 mM BTP 8.5. The crystal was transferred to a cryo-protectant consisting of well solution supplemented with 15% glycerol, mounted in a loop, and flash-cooled in liquid nitrogen.
NMR Spectroscopy:
Data Collection:2.0 Å , X-ray source: Rotating anode, FR-E superbright.
Data Processing:

References

Doyle, D.A., A. Lee, J. Lewis, E. Kim, M. Sheng, and R. MacKinnon. 1996. Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 85 : 1067-1076.
Gregorc, U., S. Ivanova, M. Thomas, E. Guccione, B. Glausinger, R. Javier, V. Turk, L. Banks, and B. Turk. 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.
Matter, K., and M.S. Balda. 2003. Signalling to and from tight junctions. Nat Rev Mol Cell Biol 4 : 225-236.