Human nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 interacting protein - Ubiquitin-like domain

Target ID:

NFATC2IP

Aliases:

FLJ14639MGC126790MGC138387

Deposition Date:

Friday 30th November 2007

Families:

Ubiquitin Related Biology

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2JXX

Authors:

Doherty R.S., Dhe-Paganon S., Fares C., Lemak A., Butler C., Srisailam S., Karra M., Yee A., Edwards A.M., Weigelt J., Arrowsmith C.H.

Site:

Toronto

ICM Datapack:

ICM Datapack

2JXX

tabs

Structure Details

The ubiquitin-like fold consists of a 5-strand mixed β-sheet that is intercalated by a 2-helix α-helical core. The Human genome encodes ~80 ubiquitin-like fold containing proteins, of which a dozen are ubiquitin-like modifiers. Of the ubiquitin-like modifiers that have been functionally characterized, the second ubiquitin-like domain of NFATC2IP is most similar to SUMO1 (28% sequence identity / 54% sequence similarity). SUMO1 is usually conjugated to a surface exposed lysine found within conserved motifs (ie. ФKXE), and is involved in transcriptional regulation and genome surveillance.

NFATC2IP is involved in the Nuclear factor of activated T-cells (NFAT) signaling cascade, which is important in immune response. The NFAT family of transcription factors (NFATc1, NFATc2, NFATc3, and NFATc4) are characterized by a Rel-homology region and an NFAT-homology region. NFATc2 interacts with NFATC2IP, and is present in the cytoplasm prior to translocating to the nucleus upon T-cell receptor stimulation. NFATc2 contains a putative SUMO interacting motif, which could be involved in the association between NFATC2IP and NFATc2. NFATc2 also contains sumoylation sites, where SUMO conjugation leads to nuclear retention, regulation of transcriptional activity and recruitment to nuclear SUMO-1 bodies.

The full length NFATC2IP protein has a highly disordered N-terminus, where Arginine methylation by PRMT modulates its interaction with NFATc2. The C-terminus of NFATC2IP contains two ubiquitin-like domains, of which we determined the solution structure of the second ubiquitin-like domain. The solution structure of the Ubiquitin-like domain of NFATC2IP reveals a large negative surface patch at the C-terminus, as well as structural conservation of 22 residues from SUMO1 with no strong correlation with characterized interactions.

Materials and Methods

NFATC2IP

PDB: 2JXX
Entry Clone Accession: NP_116204.3
Entry Clone Source: ubh72.BC021551.OBS.40K16.pOTB7
SGC Clone Accession: ubh72.342.419 (SDC088D093)
Tag: N-terminal: MGSSHHHHHHSSGLVPRGS
Host: BL21 (DE3)
Vector:pET28-MHL

Sequence:MGSSHHHHHHSSGLVPRGSTETSQQLQLRVQGKEKHQTLEVSLSRDSPLKTLMSHYEEAMGLSGRKLSFFFDGTKLSGRELPADLGMESGDLIEVWG

Growth

Medium: M9 minimal media: 100 uM ZnSO₄, 8.55 mM NaCl, 47.6 mM Na₂HPO₄, 22 mM KH₂PO_4, 100 mM MgSO₄, 2 mM biotin, 1.5 mM thiamine.HCl, 10 mM ZnSO₄, and 0.1 M CaCl₂.
Procedure:A 125 ml flask containing M9 minimal media, supplemented with ¹⁵NH₄Cl, ¹³C6-D-glucose, and 50 µg/ml kanamycin was inoculated from a glycerol stock of bacteria. The flask was shaken overnight (18 hours) at 220 rpm at 37 degC.A 2L flask containing 1000 ml minimal media supplemented with 50 µg/ml kanamycin was inoculated with the 50 ml overnight starter culture, and incubated at 37 degC to an OD600 of 1.0. Protein expression was induced with 100 µM isopropyl-thio-ß-D-galactopyranoside and the cells were incubated for overnight (15.5 hours) at 220rpm at 15 degC, and cell pellets collected by centrifugation and frozen in 50 mL Falcon tubes at -80 degC.

Purification

Procedure:
IMAC: The lysate was clarified by centrifugation for 20 min at 4 degC. The supernatant was mixed with 2 mL of Ni²⁺ affinity beads per 40 mL lysate. The mixture was incubated with mixing for 20 minutes at 4 degC. The lysate was spun at 2000 rpm for 6 minutes, and the supernatant was decanted. The remaining resin was resuspended and washed twice with lysis buffer, followed by two washes with with 5 mL of cold wash buffer. The washed resin was transferred to a gravity filter column and further washed with 2 mL of wash buffer. Samples were eluted from the resin by exposure to 5mL of elution buffer.

Buffer exchange & protein concentration: The purified protein was exchange from elution buffer into MOPS-based NMR buffer by ultracentrifugation using 2 mL concentrators with a 3,000 molecular weight cut-off (VivaSpin 2 MES) at 3000 rpm, resulting in a final volume of 300ul (final protein concentration of 0.9 mM). The concentrated protein was transferred to a 3mm NMR tube.

Residual dipolar coupling sample alignment: After data collection was performed on the unaligned sample, the purified protein was aligned by titrating 12 mg/ml Pf1 co-solvent Protease-free Phage into the NMR sample until 10 Hz proton splitting was observed.

Extraction

Procedure: The frozen cell pellet stored in a 50 ml Falcon tube obtained from 1L of culture was thawed by soaking in warm water. Each cell pellet was resuspended in 40 mL lysis buffer and lysed by sonication on ice.

Structure Determination

NMR Spectroscopy:A series of spectra (3D 1H-13C NOESY, 3D 1H-15N NOESY, 2D 1H-13C Constant Time HSQC, 3D HNCO, 3D HNCA, 3D CBCA(CO)NH, 3D HBHA(CO)NH, 3D 1H-13C Aromatic NOESY, 3D (H)CCH-TOCSY, and 3D H(C)CH-TOCSY) were generated using a 500MHz Bruker AVANCE spectrometer, a 600MHz Bruker AVANCE spectrometer and a 800MHz Bruker AVANCE spectrometer. Spectra of aligned and unaligned spectra (2D 1H-15N IPAP HSQC) were obtained using the 500MHz Bruker AVANCE spectrometer and the 800MHz Bruker AVANCE spectrometer. NMR data was processed and analyzed using Topspin, NMRPipe, NMRDraw, Sparky, Abacus, FMC-GUI, CNS, TALOS, PALES, PSVS, and WhatIF.