Human mucosa associated lymphoid tissue lymphoma translocation gene 1 (Malt1) immunoglobulin-like domain

Target ID:

MALT1

Aliases:

DKFZp434L132MLTMLT1

Deposition Date:

Thursday 22nd November 2007

Families:

Proteases

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

3BFO

Authors:

Walker, J.R., Akutsu, M., Littler, D.R., Li, Y., Arrowsmith C.H., Weigelt J., Edwards A.M., Botchkarev A., Dhe-Paganon, S.

Site:

Toronto

ICM Datapack:

ICM Datapack

3BFO

tabs

Structure Details

NF-κB regulates both innate and adaptive immune systems through transcription of various target genes[2]. Its activation is highly regulated; most notably by the upstream Iκ-B kinase (IKK) complex, which phosphorylates Iκ-B, releasing a latent nuclear localization signal in NF-κB allowing subsequent nuclear translocation and transcriptional activation. Phosphorylation of Iκ-B leads to its ubiquitylation and subsequent proteasomal degradation.

Originally identified as a translocation breakpoint in mucosa-associated lymphoid tissue lymphoma patients[1], Malt1 plays an important role in the activation of the IKK complex, but significant details are missing[2]. Known is that together with CARMA1 and BCL10, Malt1 behaves as an E3 ligase that targets the γ-subunit of the IKK complex for K63 ubiquitylation. The oligomeric form of BCL10 is believed to activate Malt1[3, 4]; interaction between these proteins is likely mediated by an immunoglobulin-like domain in Malt1[5].

To begin to better understand how Malt1 activates IKK, we included studies on the immunoglobulin-like domain and solved a 1.2 Angstrom resolution crystal structure. This structure may also hold clues for drug design in this pathway.

Materials and Methods

MALT1

PDB:3BFO

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_006776
Entry Clone Source:malt1.BC030143.MGC.AU79-A6.pOTB7
SGC Clone Accession:malt1.226.314.128B09 (SDC128B09)
Tag:N-terminal: MGSSHHHHHHSSGLVPR*GS (removed)
Host:E.coli BL21(DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvpr*GSKLQICVEPTSQKLMPGSTLVLQCVAVGSPIPHYQWFKNELPLTHETKKLYMVPYVDLEHQGTYWCHVYNDRDSQDSKKVEIIIDELNNL
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:The protein was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/mL of kanamycin at 37ºC to an OD600 of 7.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.05 mM, and incubated overnight at 15ºC. The culture was centrifuged and the cell pellets were collected and stored at -80ºC.

Purification

Procedure:
IMAC: The cleared lysate from a 2 L culture was loaded onto 3 ml TALON metal-affinity resin column (BD Biosciences) at 4ºC. The column was washed with 40 ml Wash buffer, and the protein was eluted with 10 ml Elution buffer.
Tag removal: 1 Unit of thrombin (Sigma T9681) per milligram of protein was added to the 10 mL sample, stored overnight without shaking at 4ºC.
Gel-filtration: It was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer and concentrated to 50 mg/ml by ultrafiltration using Amicon Ultra centrifugal filter with 10 kD cutoff and stored at -80 ºC.
Protein yield was 12 mg per liter of bacterial culture.

Extraction

Procedure: The cell pellet was defrosted and cells were lysed by sonication, 10 s on, 10 s off at 40% amplitude for 10 min. The lysate was cleared by centrifugation for 45 minutes at 15,500 RPM, 4°C.
Concentration:50 mg/ml
Ligand:
MassSpec:Mass-spectroscopy by LCMS shows that the product was pure and of correct molecular weight.
Crystallization:Purified protein was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (50 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 30% PEG 550-MME, 0.2 M Ammonium sulfate, and 0.1 M Sodium cacodylate at pH 6.5 in 293 Kelvin.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Akagi T, Motegi M, Tamura A, Suzuki R, Hosokawa Y, Suzuki H, Ota H, Nakamura S, Morishima Y, Taniwaki M, Seto M (1999) A novel gene, MALT1 at 18q21, is involved in t(11;18) (q21;q21) found in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue. Oncogene 18: 5785-5794
Schulze-Luehrmann J, Ghosh S (2006) Antigen-receptor signaling to nuclear factor kappa B. Immunity 25: 701-715
Matsumoto R, Wang D, Blonska M, Li H, Kobayashi M, Pappu B, Chen Y, Wang D, Lin X (2005) Phosphorylation of CARMA1 plays a critical role in T cell receptor-mediated NF-kappaB activation. Immunity 23: 575-585
Du MQ. (2007) MALT Lymphoma : Recent Advances in Aetiology and Molecular Genetics. J Clin Exp Hematop. 47: 31-42.
Lucas PC, Yonezumi M, Inohara N, McAllister-Lucas LM, Abazeed ME, Chen FF, Yamaoka S, Seto M, Nunez G. Bcl10 and MALT1, independent targets of chromosomal translocation in malt lymphoma, cooperate in a novel NF-kappa B signaling pathway. J. Biol. Chem. 2001 Jun 1;276(22):19012-9.