Human pseudouridylate synthase 7, catalytic domain

Target ID:

PUS7

Deposition Date:

Wednesday 22nd June 2016

Families:

Transferases

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

5KKP

Authors:

Dong A, Zeng H, Walker JR, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Wu H

Site:

Toronto

5KKP

tabs

Structure Details

RNA modifications are emerging as an epigenetic pathway for regulation of gene expression. More than 100 types of post-transcriptional modifications have been identified in cellular RNAs. Pseudouridine is the most abundant modified base in cells. It has been found in tRNA, rRNA, snRNA and other non-coding RNAs. These modifications are essential for biological functions such as spliceosome biogenesis. It is also required for the progression of the apoptotic signal through intrinsic mitochondrial cell death. Recent studies have shown detection of pseudouridine in yeast and human transcriptome and the level of pseudouridylation in mRNA can be induced by environmental cues such as stress, but the functional role of pseudouridylation in mRNA is still unclear. Pseudouridine has been linked to human diseases. An increased level of oxidized pseudouridine has been associated with neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. Mutations in pseudouridine synthase box H/ACA RNP have been linked to the X-linked form of the bone marrow failure syndrome dyskeratosis congenita. Formation of pseudouridine involves the detachment, flipping and reattachment of U base to the ribose. This process is catalyzed by a family of pseudouridine synthases.

Pseudouridylate Synthase 7 (Pus7) belongs to the TruD sub-family of pseudouridine synthases. In yeast, Pus7p is responsible for modification at position U35 in Saccharomyces cerevisiae U2 snRNA, U13 in tRNA and U35 in pre-tRNATyr. Pseudouridination at U2 in snRNA is important for high splicing efficiency in yeast. We have solved the crystal structure of human Pus7 at 2.26 Å resolution. Pus7 adopts a V-shaped conformation that is conserved in TruD sub-family of pseudouridine synthases. The structure can be divided into two distinct domains: the catalytic domain and the insertion domain. The two domains are connected at the bottom of the V shape. The catalytic domain is characterized by an eight-stranded β sheet core. The core sheet is also decorated by helices and loops on one side, surrounding the catalytic cleft. The insertion domain features a mixed α/β structure.

Materials and Methods

PUS7

PDB:5KKP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC011396
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: 6XHis-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gESFADMMKHGLTEADVGITKFVSSHQGFSGILKERYSDFVVHEIGKDGRISHLNDLSIPVDEEDPSEDIFTVLTAEEKQRLEELQLFKNKETSVAIEVIEDTKEKRTIIHQAIKSLFPGLETKTEDREGKKYIVAYHAAGKKALANPRKHSWPKSRGSYCHFVLYKENKDTMDAINVLSKYLRVKPNIFSYMGTKDKRAITVQEIAVLKITAQRLAHLNKCLMNFKLGNFSYQKNPLKLGELQGNHFTVVLRNITGTDDQVQQAMNSLKEIGFINYYGMQRFGTTAVPTYQVGRAILQNSWTEVMDLILKPRSGAEKGYLVKCREEWAKTKDPTAALRKLPVKRCVEGQLLRGLSKYGMKNIVSAFGIIPRNNRLMYIHSYQSYVWNNMVSKRIEDYGLKPVPGDLVLKGATATYIEEDDVNNYSIHDVVMPLPGFDVIYPKHKIQEAYREMLTADNLDIDNMRHKIRDYSLSGAYRKIIIRPQNVSWEVVAYDDPKIPLFNTDVDNLEGKTPPVFASEGKYRALKMDFSLPPSTYATMAIREVLKMDTSIKNQTQLNTTWLR
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:PUS7 protein was expressed in E.coli BL21 (DE3) codon plus RIL in M9 minimum medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37oC to an OD600 of 0.8 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, in the presence of 50 mg/L of SeMet and incubated overnight at 15oC.

Purification

Buffers
Procedure
The crude extract was cleared by centrifugation. The lysate was loaded onto 5 ml HiTrap column (GE Healthcare), charged with Ni2+. The column was washed with 10 CV of 20 mM HEPES pH 7.4, containing 500 mM NaCl, 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded on Superdex200 column (26x60) (GE Healthcare), equilibrated with 20 mM PIPES, pH 6.5, 250 mM NaCl. The fractions containing PUS7 were pooled and TEV protease was added to remove His-tag. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (GE Healthcare), equilibrated with buffer containing 20 mM PIPES, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20 CV).

Extraction

Buffers
Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80˚C. For purification, the cell paste was thawed and resuspended in lysis buffer buffer (50 mM HEPES, pH 7.4, 0.5 M NaCl, 5 mM imidazol, 2 mM β-mercaptoethanol, 5% glycerol) with protease inhibitor (1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:24.7 mg/mL - Enzymatic treatment: TEV
Ligand
MassSpec:The expected mass for PUS7 is 64415 Da, measured mass for SeMet protein is 65214 Da.
Crystallization:Purified PUS7 protein (10.1 mg/mL) was crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 μl of the protein solution with 1 μl of the reservoir solution containing 20% PEG3350, 0.2 M sodium formate, pH 7.0
NMR Spectroscopy:
Data Collection:
Data Processing: