Human helicase-like transcription factor, HIRAN domain

Target ID:

HLTF

Deposition Date:

Wednesday 28th July 2010

Families:

Miscellaneous

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

Superceded by 5K5F

Authors:

Bezsonova I, Neculai D, Weigelt J, Bountra C, Edwards AM, Arrowsmith CH, Dhe-Paganon S

Site:

Toronto

ICM Datapack:

ICM Datapack

2L1I

tabs

Structure Details

Defects in the ability to properly respond to and repair DNA damage promote genomic instability and cancer in higher-order eukaryotes. It has been shown that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role (Blastyak et al.). HLTF has also been implicated in transcription regulation (Sheridan et al., 1995). Interestingly, the gene encoding HLTF is frequently subjected to hypermethylation in human cancers (Moinova et al., 2002).

HLTF (helicase-like transcription factor) domain organization includes a HIRAN domain, the seven motifs found in SWI/SNF ATPase and a RING domain characteristic of E3 ubiquitin ligases (MacKay et al., 2009). There is no HIRAN domain structural homologue it has been predicted to bind DNA (Iyer et al., 2006). In this work we present the NMR structure of the HIRAN domain revealing a unique fold with two positively charged surfaces that could be implicated in binding to DNA The HIRAN domain of HLTF has a unique fold consisting of five β-strands, two α -helices, and two 3-residue 310 helical turns, arranged in order β1, β4, 3101, β5, α1, β3, 3102, β2 α1. The strands β1 β2 β3 β4 β5 form a single β-sheet.

Materials and Methods

HLTF

PDB:2L1I

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:Mammalian Gene Collection cDNA template
Entry Clone Source:hltf.BC044659.MGC.AT70-E3.pCMV-SPORT6
SGC Clone Accession:hltf.52.171.179F03 (SDC179F03)
Tag:MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 CodonPlus

Construct

Prelude:
Sequence:MGSSHHHHHHSSGLVPRGSDEEVDSVLFGSLRGHVVGLRYYTGVVNNNEMVALQRDPNNPYDKNAIKVNNVNGNQVGHLKKELAGALAYIMDNKLAQIEGVVPFGANNAFTMPLHMTFWGKEENRKAVSDQLKKHGFKL
Vector:pET28a-LIC vector (GenBank, EF442785)

Growth

Medium:2 L of doubly labeled minimal media
Antibiotics:
Procedure:Competent E.coli BL21 CodonPlus cells were grown in 2 L of doubly labeled minimal media at 37 °C to OD₆₀₀=1.0, then cells were induced with 1mM IPTG for 16 hrs at 15 °C.

Purification

Procedure
Cleared cell lysate in lysis buffer was loaded onto a 5 mL TALON metal-affinity resin column equilibrated in wash buffer at 4 °C temperature. The column was washed with 50 mL of wash buffer and the protein was eluted with 10 mL of elution buffer. Upon addition of 1 unit of Thrombin protease, 2 mM beta-mercaptoethanol, the eluant was incubated for 10 hrs at 4 °C. The protein was further purified by gel filtration on a HighLoad Superdex 75 column equilibrated with gel-filtration buffer. Fractions containing protein were pooled and concentrated using Amicon Ultra centrifugal filter with 3kD cutoff membrane to a final concentration of 16 mg/ml. Protein yield was 7 mg per liter of bacterial culture. Coomassie-stained SDS-PAGE showed that the ¹⁵N,¹³C labeled product was pure and mass-spectroscopy by LCMS (Agilent 1100 Series) showed that the purified protein had a molecular weight corresponding to 14266 g/mol.

Extraction

Procedure

Concentration:
Ligand
MassSpec:
Crystallization:
NMR Spectroscopy:NMR analyses were performed using a Bruker AVANCE 600 or 800 spectrometer at 298K. NMR samples contained 1.6 mM ¹⁵N, ¹³C-labeled hltf.52.171 in 20 mM sodium phosphate pH 6.3 and 100 mM NaCl with 10% D₂O. The 3D spectra were measured with nonuniform sampling (NUS) of the indirect dimensions (down to 30% sampling) and reconstructed using multidimensional decomposition (MDD) using MDDNMR (Jaravine and Orekhov, 2006). All spectra were processed using NMRPipe/NMRDraw (Delaglio et al., 1995) and analyzed using Sparky (Goddard, 2008). Backbone assignments were obtained using standard triple-resonance experiments (Bax and Grzesiek, 1993). Aliphatic side-chain assignments were obtained from 3D HCCH-TOCSY, and aromatic side-chain assignments were obtained from 2D (Hβ)Cβ(CγCδ)Hδ and (Hβ)Cβ(CγCδCε)Hε experiments (Yamazaki et al., 1993). Distance restraints, derived from a ¹⁵N- and ¹³C-edited NOESY-HSQC (using crossrelaxation mixing time of 100 ms) and dihedral angle restraints, predicted from chemical shifts with the software TALOS (Cornilescu et al., 1999) and (Jaravine and Orekhov, 2006), were used as input for combined automated NOE assignment and structure calculation with the program CYANA (Guntert, 2004).
Data Collection:
Data Processing:The coordinates and structure factors have been deposited 2010.07.28 into the RCSB PDB database with ID code 2L1I with the following authors: Irina Bezsonova, Dante Neculai, Johan Weigelt, Chas Bountra, Aled M. Edwards, Cheryl H. Arrowsmith, and Sirano Dhe-Paganon.

References

Bax, A., and Grzesiek, S. (1993). Methodological Advances in Protein Nmr. Accounts of Chemical Research 26, 131-138.
Blastyak, A., Hajdu, I., Unk, I., and Haracska, L. Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA. Mol Cell Biol 30, 684-693.
Cornilescu, G., Delaglio, F., and Bax, A. (1999). Protein backbone angle restraints from searching a database for chemical shift and sequence homology. Journal of biomolecular NMR 13, 289-302.
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. Journal of biomolecular NMR 6, 277-293.
Goddard, T.D., and Kneller, D.G. (2008). SPARKY 3. University Of California, San Francisco.
Guntert, P. (2004). Automated NMR structure calculation with CYANA. Methods in molecular biology. Clifton, NJ 278, 353-378.
Iyer, L.M., Babu, M.M., and Aravind, L. (2006). The HIRAN domain and recruitment of chromatin remodeling and repair activities to damaged DNA. Cell cycle (Georgetown, Tex 5, 775-782.
Jaravine, V.A., and Orekhov, V.Y. (2006). Targeted acquisition for real-time NMR spectroscopy. Journal of the American Chemical Society 128, 13421-13426.
MacKay, C., Toth, R., and Rouse, J. (2009). Biochemical characterisation of the SWI/SNF family member HLTF. Biochemical and biophysical research communications 390, 187-191.
Moinova, H.R., Chen, W.D., Shen, L., Smiraglia, D., Olechnowicz, J., Ravi, L., Kasturi, L., Myeroff, L., Plass, C., Parsons, R., et al. (2002). HLTF gene silencing in human colon cancer. Proceedings of the National Academy of Sciences of the United States of America 99, 4562-4567.
Sheridan, P.L., Schorpp, M., Voz, M.L., and Jones, K.A. (1995). Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter. The Journal of biological chemistry 270, 4575-4587.
Yamazaki, T., Formankay, J.D., and Kay, L.E. (1993). 2-Dimensional Nmr Experiments for Correlating C-13-Beta and H-1-Delta/Epsilon Chemical-Shifts of Aromatic Residues in C-13-Labeled Proteins Via Scalar Couplings. Journal of the American Chemical Society 115, 11054-11055.