Human mucosa associated lymphoid tissue lymphoma translocation protein 1 isoform A - Death domain

Target ID:

MALT1

Aliases:

DKFZp434L132MLTMLT1

Deposition Date:

Tuesday 28th February 2006

Families:

Proteases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2G7R

Authors:

J.R.WALKER,L.WYBENGA-GROOT,E.M.NEWMAN,P.J.FINERTY JR.,C.BUTLER-COLE,J.WEIGELT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2G7R

tabs

Structure Details

Much of our mucosal linings, such as the digestive tract, bronchial passages and vaginal canal, contain resident lymphoid cells that protect the body from invading bacteria and virus.
Cancer of mucosal-associated lymphoid tissue
(MALToma) occurs in 7% of all Non-Hodgkinâ€™s Lymphomas or in about 3000 US individuals per year.
Genetic translocation events are often a cause of this disease, usually involving the activation of proteins MALT1 and BCL10, which are upstream activators of the inflammatory NKFB pathways.
A common non- genetic cause of Gastric MALToma is Helicobacter pylori infection.
Constitutive activation of NFKB leads to excessive proliferation of lymphocytes and their resistance to apoptosis.
To be able to provide better therapeutics for this disease, a deeper understanding of the mechanism of
MALT1- and BCL10-mediated activation of NFKB is sought.

MALT1 and BCL10 appear to work in concert to activate the IKK complex, which consists of a serine/threonine kinase that phosphorylates IkB for proteasomal degradation and release of NFKB for nuclear entry/activation.
The immunoglobulin-like domains of MALT1 recognize BCL10, which is a target of T cell Receptor activation.
MALT1â€™s death domain is associated with caspase 8 and cFLIP activation, which are upregulated by CD95/FAS/TNF receptor.
How MALT1 activates the IKK complex is not known. And what is the role of the catalytic deubiquitinylase domain?

In order to determine the mechanism of lymphocyte activation and regulation, we are determining the first high resolution crystal structures of MALT1, including the amino-terminal death domain.
Our structure shows a canonical fold but with the last two carboxy terminal helices forming a continous and extended conformation.
This unique feature could be the basis of MALT1's protein/protein binding specificity.
Structures of the caspase-like domain of
MALT1 may also lay the groundwork for the discovery and development of pharmacotherapeutics directed towards MALTomas.

Materials and Methods

MALT1-DD

PDB:2G7R

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:malt1.BC030143.MGC.AU79-A6.pOTB7
Entry Clone Source:
SGC Clone Accession:malt1.029.126; plate SDC34H07
Tag:MGSSHHHHHHSSGLVPRGS
Host:E. coli. BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsTLNRLREPLLRRLSELLDQAPEGRGWRRLAELAGSRGRLRLSCLDLEQCSLKVLEPEGSPSLCLLKLMGEKGCTVTELSDFLQAMEHTEVLQLLSPPG
Vector:pET28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:Media bottles (2 L) containing TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ ml kanamycin and 600 ul antifoam 204 (Sigma A-8311) were inoculated with 50-100 ml of the overnight LB culture each. With sterilized cap/sparger (Fisher 11-138B) assemblies, bottles were placed into a circulating water bath set at 37 degC. Temperature was reduced to 15oC one hour prior to induction at OD(600) between 4 and 8 with 100 uM isopropyl-thio-B-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 degC.

Purification

Procedure
Three milliliters of TALON metal-affinity resin (BD Bioscience) was mixed for 2 hours at 4 degC with 40 mL lysate, centrifuged for 3 minutes (SX4750 rotor, Allegra X-12R, Beckman Coulter), and decanted. Beads were transferred into a 25 mL Econo-Column (Bio-Rad 732-1010) and washed with 5 x 12 mL Wash buffer. Samples are eluted with 2-3 column volumes of Elution buffer. EDTA was added up to 1 mM to each protein sample followed by 2 mM DTT. Samples were gel-filtered (XK 16x65 packed with HighLoad Superdex 200 resin, GE Healthcare) using an AKTAxpress (18-6645-05, GE Healthcare) at a flow rate of 1 mL/min Gel-filtration buffer, and 2 mL fractions were collected in 96-well plates. Fractions containing protein were pooled and centrifuged through concentrators with 5,000 kDa cut-off (Amicon Ultra-15, UFC900524, Millipore) for 45 minutes at 3750 rpm.

Extraction

Procedure
Frozen cell pellets from 2L cultures contained in bags (Beckman 369256) were thawed by soaking in warm water, resuspended in 25-40 mL Lysis buffer, and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds. Cell lysis was accomplished with a Microfluidizer Processor M-110EH (Microfluidics) at 18,000-20,000 psi with ice cooling. Aliquots of PMSF were added (60-80 µL of 100 mM stock) prior to centrifugation at 10°C for 30 minutes (JA25.50 rotor at 23,000 rpm). Supernatants were decanted into sterile 50-ml polypropylene conical tubes.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were grown at 298 K using the hanging drop method by mixing equal volumes of 1.0 M K/Na tartrate, 0.1 M MES pH 6.0 and 10 mg/mL protein. Crystals did not require cryoprotection before freezing in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing: