Human malignant T cell amplified sequence 1

Target ID:

MCTS1

Deposition Date:

Thursday 24th March 2011

Families:

Miscellaneous

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

3R90

Authors:

Hong B, Dimov S, Tempel W, Tong Y, Wernimont AK, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Park H

Site:

Toronto

3R90

tabs

Structure Details

Genomic alteration through gene amplification has been recognized as an important event in
malignant lymphomagenesis. Multiple copies in T-cell lymphoma-1 (MCT-1) is an oncogene
that was originally identified in a certain lymphoma cell line, where it was found amplified.
Increased levels of MCT-1 protein were observed in a wide array of human lymphoma cells
including aggressive non-Hodgkin's lymphomas, and lymphoid cell lines overexpressing MCT-
1 displayed increased growth rates and resistance to apoptosis. These findings suggest that the
elevated level of MCT-1 protein is linked to cell transformation and proliferation, thereby being
potentially implicated in human lymphomagenesis, although the exact molecular mechanism
underlying this process remains elusive. MCT-1 is composed of 181 amino acid residues and
contains a pseudouridine synthase and archaeosine transglycosylase (PUA) domain at its C-
terminus. In addition, MCT-1 protein recruits the density regulated protein (DENR/DRP), which
contains an SUI1 domain involved in initiation site selection during translation.

We determined the crystal structure of apo MCT-1 at 1.7 Å resolution using the surface-entropy-
reduced (SER) method (E137A, K139A, Q140A, referred to as MCT-1SER). The overall structure
of MCT-1SER showed two globular and compact domains (designated herein as "N" and "C")
separated by two short linker peptides, and each domain was composed of a mixture of four α-
helices and five β-sheets, respectively. N-domain (residues 1-92) has a five-stranded antiparallel
β-sheet with three α-helices on one side and two α-helices on the other side. As anticipated,
the C-domain (residues 93-181) of MCT-1SER adopted a highly conserved PUA fold, which
has a central pseudobarrel. Both apical sides of the pseudobarrel were flanked by two helices,
respectively.

Materials and Methods

MCTS1

PDB:3R90

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:Q9ULC4 / BC001013
Entry Clone Source:MGC AU9-E2
SGC Clone Accession:HPC0AA-D07
Tag:C-terminal his tag (AHHHHHH)
Host:BL21(DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:MFKKFDEKENVSNCIQLKTSVIKGIKNQLIEQFPGIEPWLNQIMPKKDPVKIVRCHEHIEILTVNGELLFFRQREGPFYPTLRLLHKYPFILPHQQVDKGAIKFVLSGANIMCPGLTSPGAKLYPAAVDTIVAIMAAGAAHALCVGVMKMSAEDIEKVNKGIGIENIHYLNDGLWHMKTYKahhhhhh Underlined letters : susbstiuted residues E137A / K139A / Q140A
Vector:pNIC-CH

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 40~50 mL of overnightculture grown in Luria-Bertani medium into a 2 L of Terrific Broth medium in the presence of50 µg/mL kanamycin and 30 µg/mL chloramphenicol at 37 °C. When OD600 reached ~3.0, thetemperature of the medium was lowered to 17 °C and the culutre was induced with 0.5 mM IPTG.The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen andstored at -80 °C.

Purification

Procedure
The cell lysate was then centrifuged to remove insoluble material, and the supernatant was loadedonto a DEAE-cellulose (DE52, Whatman, MA, USA) anion-exchange resin followed by a nickel-NTA agarose column (Qiagen, MD, USA). Bound proteins were eluted with a elution. The elutedsample was then dialyzed and subjected to cation-exchange chromatography using a HiTrapSP column (GE Healthcare, NJ, USA) previously equilibrated with the same buffer used fordialysis. Protein was eluted with a linear gradient of 0-500 mM NaCl, and further purified by sizeexclusion chromatography on a Superdex 75 16/60 column (GE Healthcare, NJ, USA). The peakfractions containing MCTS-1 protein were pooled, concentrated using amicon centrifugal filterand stored at -80 °C before crystallization.

Extraction

Procedure
Frozen cells were thawed and suspended in 150 mL the binding bufferand supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 2 µL (SigmaCatalog # E1014, 250U/µL) benzonase, and lysed using a Microfluidizer at 16,000 psi.
Concentration:30 mg/mL
Ligand
MassSpec:
Crystallization:Native crystals : 2.5M AmSO4; Bis-Tris propane pH 7.0
SeMet-derivatized crystals : 2.5M AmSO4; Bis-Tris propane pH7.0; 20% PEG3350
NMR Spectroscopy:
Data Collection:
Data Processing: