Human tight junction protein 3 (zona occludens 3), kinase domain

Target ID:

TJP3

Deposition Date:

Wednesday 28th October 2009

Families:

Non-protein Kinase

Structure type:

apo

Download Coordinates:

3KFV

Authors:

Tong Y, Nedyalkova L, Tempel W, Zhong N, Guan X, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park HW

Site:

Toronto

ICM Datapack:

ICM Datapack

3KFV

tabs

Structure Details

Tight junction (TJ) is composed of a branching network of sealing strands which links adjacent epithelial or endothelial cells and plays a barrier role by regulating the passage of ions and molecules through the paracellular pathway[1,2]. TJP3/ZO-3 (tight junction protein 3 or zonula occludens 3) is a TJ cytoplasmic plaque protein. TJP3 consists of three PDZ domains (PDZ1-PDZ3), one SH3 domain, and one guanylate kinase (GK) domain, and belongs to the membrane-associated guanylate kinase (MAGUK) family. The PDZ2 domain of TJP3 binding to that of TJP1 to form a stable complex[3]. The PDZ1 domain of TJP3 binds to a transmembrane TJ protein claudin, localizing it at the TJ region to carry out function for cell-cell adhesion[4]. The GK domain of TJP3 is homologous to the enzyme that catalyzes the conversion of GMP to GDP at the expense of ATP. But the TJP proteins are predicted to bind neither GMP nor ATP and thus are enzymatically inactive[5].

Here, we solved the crystal structure of the apo-form of a fragment of TJP3 containing the SH3 and GK domains. The SH3 domain adopts a typical α/β fold, while the GK domain shows an incomplete kinase fold, where the N-lobe is missing, thus collapsing the cleft between the N- and C-lobes. Since the cleft forms the nucleotide binding site (i.e.,g GMP and ATP) in authentic kinases, the TJP3 structure explains that the enzymatic inactivation of TJP3 is due to GMP and ATP being unable to bind to the collapsed cleft.

Materials and Methods

TJP3

PDB:3KFV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:SGC cDNA library: 20-E5:TJP3
Entry Clone Source:Codon Devices Synthesized
SGC Clone Accession:HPC092-C03
Tag:C-terminal: HHHHHH
Host:BL21-V2R-pRARE2

Construct

Prelude:TJP3:G508-L808:cH6
Tag not removed
Sequence:mGDSFYIRTHFELEPSPPSGLGFTRGDVFHVLDTLHPGPGQSHARGGHWLAVRMGRDLREQERGIIPNQSRAEQLASLEAAQRAVGVGPGSSAGSNARAEFWRLRGLRRGAKKTTQRSREDLSALTRQGRYPPYERVVLREASFKRPVVILGPVADIAMQKLTAEMPDQFEIAETVSRTDSPSKIIKLDTVRVIAEKDKHALLDVTPSAIERLNYVQYYPIVVFFIPESRPALKALRQWLAPASRRSTRRLYAQAQKLRKHSSHLFTATIPLNGTSDTWYQELKAIIREQQTRPIWTAEDQLhhhhhh
Vector:pET28a-LIC-CHis (NCBI GI:145307000) Digested with NcoI and BseRI

Growth

Medium:
Antibiotics:
Procedure:LEX Bubbling. Selenomethionine labeling of the target protein was carried out using the M9 SeMet growth media kit from Medicilon (Cat. MD045004) following manufacturer's instructions. Briefly, 50 mL overnight E. coli Luria-Bertani culture was inoculated into a 2 L of M9 medium in the presence of NIAAC, thiamine and Vitamin B12 mix, fifteen mineral supplements, in the presence of 50 µg/mL kanamycin and 35 µg/mL chloramphenicol at 37 °C. When OD₆₀₀ reached ~1.2, 40 mL stock solution containing inhibitory amino acid cocktail (IAAC) and Se-Met were added to the M9 culture and the temperature of the medium was lowered to 18 °C. After fifteen minutes, the culture was induced with IPTG at a 1 mM final concentration. The cells were allowed to grow overnight before being 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 supernatants were mixed with 5 mL 50% flurry of TalonÂ Cobalt beads and incubated at 4 C on rotary shaker for one hour. The mixture was then centrifuged at 2300 rpm for 5 min and the supernant was discarded. The beads were then washed with washing buffers containing 30 mM and 75 mM imidazole, and finally the bound protein was eluted using the elution buffer. The elutant was collected and further purified on a Superdex-200 gel filtration column pre-equilibrated with gel filtration buffer. Fractions containing the protein were collected and concentrated with Amicon Ultra-15 centrifugal filter. The purity of the preparation is tested by SDS-PAGE to be greater than 95%.

Extraction

Procedure
Frozen cells from 2L M9 culture were thawed and resuspended in 150 mL extraction buffer 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 microfluidizer at 15,000 PSI.
Concentration:15.2 mg/mL
Ligand
MassSpec:Se-Met protein expected 34945, measured 36596
Crystallization:Crystallization was setup in sitting drops with Red Wings and SGC-I screens initially. Diffracting crystals were found from condition SGC-G4. Optimization was carried out in hanging drop setup. Crystal used for structure determination was grown in 5% MPD, 1.4 M (NH₄)₂SO₄, 0.1 M HEPES buffer at pH 7.5, with 10 mM ATP in the protein stock solution, in hanging drop setup. Paratone was used as cryoprotectant. Crystals grow to a mountable size within 1 week.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

J.M. Anderson and M. Cereijido (2001) Introduction: evolution of ideas on the tight junction, CRC Press, Boca Raton pp. 1-18.
Cereijido M, González-Mariscal L, Contreras RG, Gallardo JM, García-Villegas R, Valdeacute;s J (1993) The making of a tight junction. J Cell Sci 17:127-132
Wittchen ES, Haskins J, Stevenson BR (1999) Protein interactions at the tight junction: actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem 274:35179-35185
Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S (1999) Direct binding of three tight junctionassociated MAGUKs, ZO-1, ZO-2 and ZO-3, with the COOH termini of claudins. J Cell Biol 147:1351-1363
González-Mariscal L, Betanzos A, Avila-Flores A (2000) MAGUK proteins: structure and role in the tight junction. Semin Cell Dev Biol 11(4):315-24.