Human mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli), ATPase-like domain, in complex with ATP

Target ID:

MLH1

Deposition Date:

Monday 31st May 2010

Families:

Miscellaneous

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

Superceded by 4P7A

Authors:

Lam R, Zeng H, Loppnau P, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J, Wu H

Site:

Toronto

ICM Datapack:

ICM Datapack

3NA3

tabs

Structure Details

MLH1 is a human homolog of E. coli DNA mismatch repair protein MutL. This gene is frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). MLH1 forms a heterodimer with mismatch repair endonuclease PMS2. This heterodimer (MutL alpha) is a component of the post-replication DNA mismatch repair system (MMR) and interacts with MSH2 bound to mismatched bases. This interaction brings together MutL and MutS (MSH2-MSH6) to form the MutL-MutH-heteroduplex and leads to the activation of endonuclease activity of PMS2, which initiates the repair process. MutL alpha also interacts physically with the clamp loader subunits of DNA polymerase III, suggesting its role in recruiting DNA polymerase III to the site of MMR and in DNA damage signaling.

We have solved the crystal structure of human MLH1 protein in complex with ATP at 2.5 Å resolution.

Materials and Methods

MLH1

PDB:3NA3

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI: 4557757
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQG
Host:E.coli BL21 (DE3) V2R-pRARE

Construct

Prelude:
Sequence:gMSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSS
Vector:pET28-MHL

Growth

Medium:Terrific Broth medium in the presence of 50 μg/ml of kanamycin
Antibiotics:
Procedure:MLH1 was expressed in E.coli BL21 (DE3) V2R-pRARE in Terrific Broth medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37°C to an OD₆₀₀ of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 15°C.

Purification

Procedure
The lysate was loaded onto 5 mL HiTrap column (Amersham Biosciences), charged with Ni²⁺. The column was washed with 10 CV of wash buffer and the protein was eluted with elution buffer. The protein was then loaded on to a Superdex200 (60X60, Amersham Biosciences) column equilibrated in 20 mM PIPES, pH 6.5 buffer containing 250 mM NaCl. TEV protease was added to combined fractions containing MLH1. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM PIPES, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 23 mg of the protein per 1L of culture.

Enzymatic Treatment: TEV cleavage

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80oC. For the purification the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:16.6 mg/ml
Ligand
MassSpec:Expected MW is 37675.00Da, measured mass is 37676.8 Da.
Crystallization:Purified MLH1 was complexed with ADP and crystallized using sitting drop vapor diffusion method at 20°C by mixing 1 µl of the protein solution (8 mg/ml) with 1 µl of the reservoir solution containing 20% PEG 4,000, 10% isopropanel, 0.1 M HEPES, pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing: