|Home | About | Journals | Submit | Contact Us | Français|
Human UHRF1 belongs to the unique mammalian family of proteins which contain a SET- and RING finger-associated (SRA) domain. This 180-residue domain has been reported to play key roles in the functions of the protein. It allows UHRF1 to bind methylated DNA, histone deacetylase 1 and DNA methyltransferase 1, suggesting a bridge between DNA methylation and the histone code. No structural data is available for any SRA domain. Native and SeMet-labelled SRA domains of human UHRF1 were overexpressed in Escherichia coli cells, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. A complete MAD data set was collected to 2.2 Å resolution at 100 K. Crystals of the SeMet-labelled protein belonged to the trigonal space group P3221, with unit-cell parameters a = b = 53.78, c = 162.05 Å.
In all eukaryotes, DNA is compacted in the nucleus as chromatin, the building block of which is the nucleosome core particle. It consists of 146 base pairs wrapped around an octamer of core histones formed by two copies each of histones H2A, H2B, H3 and H4. The positively charged amino-terminal tail of each histone protrudes from the nucleosome core particle and is subjected to post-translational modifications that modulate the compactness of the chromatin. Jenuwein and coworkers proposed that specific combinations of modifications of histone-tail residues (namely the ‘histone code’) modulate gene expression (Jenuwein & Allis, 2001 ). These covalent modifications act as recognition signals that direct the binding of nonhistone proteins and determine the active or repressed transcriptional state of chromatin (de la Cruz et al., 2005 ).
The human and mouse UHRF1 proteins (also called ICBP90 and NP95) are the best characterized members of the UHRF family. They contain, in the following order, a ubiquitin-like domain, a PHD finger, an SRA domain and a RING finger, which confers E3 ubiquitin ligase activity. In addition to their critical role in cell proliferation, these nuclear proteins appear to participate in epigenetic events. They play a role in maintaining DNA methylation through complex formation with DNA methyltransferase 1 (Achour et al., 2008 ; Bostick et al., 2007 ; Sharif et al., 2007 ). Mouse UHRF1 has been shown to be a new partner for pericentric heterochromatin replication in a process that involves the regulation of histone H4 deacylation (Papait et al., 2007 , 2008 ).
The SRA domain is mainly found in plant and mammalian proteins. In Arabidopsis thaliana it is found in histone methyltransferases, where it is involved in directing DNA methylation to target sequences (Naumann et al., 2005 ). In mammals it has only been identified to date in the UHRF family, in which it displays several functions. Initially, it was characterized as a new methyl-DNA binding domain (Woo et al., 2007 ; Johnson et al., 2007 ), with a preference for binding hemimethylated CpG sites in the case of UHRF1 (Bostick et al., 2007 ). It has also been implicated in the binding of UHRF1 to histone deacetylase 1 (Unoki et al., 2004 ) and it has recently been shown that the SRA domain promotes the binding affinity of human UHRF1 to K9-methylated histone H3 (Karagianni et al., 2008 ).
No structural data have been published to date for any SRA domain and here we present the first report of the expression, purification, crystallization and preliminary X-ray analysis of the SRA domain of the human UHRF1 protein (hUHRF1).
The coding sequence for the SRA domain of hUHRF1 (SRA; residues 408–643) was cloned using Gateway technology into the pHGWA expression vector (Busso et al., 2005 ), which provides an N-terminal hexahistidine tag plus a TEV cleavage site. After TEV digestion, three residues (GHM) remained at the N-terminus of the SRA protein.
Escherichia coli BL21-pLysS(DE3) cells harbouring the SRA expression plasmid were cultured in LB medium containing 100 µg ml−1 ampicillin and 35 µg ml−1 chloramphenicol and incubated at 310 K until the OD600 reached 0.35. The temperature was shifted to 293 K for 30 min and isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. Growth was continued for 5 h, after which the cells were harvested by centrifugation and lysed by sonication in buffer A (30 mM Tris–HCl pH 7, 150 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol and 1 mM PMSF) at 277 K. The lysate was clarified by centrifugation at 12 000g for 90 min at 277 K and was loaded onto a 5 ml Ni2+-loaded HiTrap Chelating HP column (GE Healthcare). The SRA protein was eluted with a linear gradient of imidazole from 10 to 300 mM in buffer A. The SRA fractions were identified by SDS–PAGE, pooled and dialyzed overnight against buffer A. The dialyzed protein was digested with recombinant tobacco etch virus (rTEV) protease for 72 h at 277 K at a ratio of 1 mg rTEV protease per 40 mg protein and the reaction mixture was applied onto an Ni2+ column. The SRA protein was recovered in the flowthrough fractions, concentrated and loaded onto a size-exclusion chromatography column (Superdex 75 HR 16/50, Amersham Biosciences) equilibrated with 25 mM Tris–HCl pH 7, 150 mM NaCl, 0.5 mM PMSF, 5 mM β-mercaptoethanol (hereafter referred to as high-salt buffer).
For the production of selenomethionine (SeMet) labelled protein, the methionine-auxotroph strain B834 (DE3) harbouring both the SRA expression plasmid and the pLysS plasmid was cultured in minimal media M9 containing 17 amino acids, selenomethionine (50 µg ml−1), ampicillin and chloramphenicol (LeMaster & Richards, 1985 ). The conditions for growing the cells and expressing and purifying the SeMet-labelled protein are identical to those described above for the native protein. Electrospray ionization mass spectrometry of the purified SeMet-labelled SRA protein confirmed the substitution of the three methionine residues by selenomethionine (100% incorporation).
Prior to dynamic light-scattering (DLS) experiments, the protein samples were centrifuged for 10 min at 14 000g. DLS studies were performed using a DynaPro801 DLS instrument (Protein Solutions Inc.). DYNAMICS v.5.25.44 software was used in data collection and analysis. For each experiment, 20 scans were collected at 293 K.
Small-angle X-ray scattering (SAXS) measurements were performed on DESY beamline X33 (EMBL Hamburg, Germany). The SRA samples were used at a concentration of 3 mg ml−1. Each protein sample was exposed for 120 s at 288 K after the addition of 2 mM DTT to eliminate the free radicals formed in solution under X-ray irradiation. Values obtained for buffer solutions without protein were subtracted from the protein data using PRIMUS (Konarev et al., 2003 ). The corrected profiles are shown in Fig. 1 .
The SRA protein was concentrated to 8 mg ml−1 in high-salt buffer. Initial crystallization screens were set up with a Mosquito nanodrop robot (Molecular Dimensions) using commercially available kits. Drops consisting of 100 nl protein solution and 100 nl reservoir solution were equilibrated against 80 µl reservoir solution. The initial crystallization condition for SRA was obtained using solution No. 10 of the JCSG screen (Page & Stevens, 2004 ). After optimization, crystals appeared at 295 K in two or three days in hanging drops composed of 1 µl protein solution at 8 mg ml−1 and 1 µl reservoir containing 22–26% unbuffered PEG 3350 solution equilibrated against 1 ml reservoir. Using protein dialyzed against 10 mM Tris–HCl pH 7, 20 mM NaCl, 5 mM MgCl2, 0.1 mM TCEP (hereafter referred to as low-salt buffer), crystals appeared in 1 d with the same drop setup and a reservoir containing 14–19% unbuffered PEG 3350 solution. The SeMet-labelled SRA protein behaved similarly to the native protein and crystals of SeMet-labelled protein were obtained under the same conditions. Typical crystal dimensions were 200 × 200 × 400 µm (Fig. 2 ).
Prior to X-ray analysis, crystals of SeMet-labelled SRA protein were soaked in a cryoprotectant solution composed of 50% reservoir and 50% ethanol by volume for 10 s, which was followed by a quick transfer to paraffin oil prior to flash-cooling in liquid nitrogen.
A complete multiple anomalous dispersion (MAD) data set was collected at 100 K from a single SeMet crystal on the European Synchrotron Radiation Facility (ESRF, Grenoble, France) beamline ID14-EH4 equipped with a Q315 ADSC detector. The fluorescence spectrum recorded from the frozen crystal was used to select the wavelength of the Se K absorption edge at the peak (λ = 0.9791 Å, maximum of f′′), at the inflection (λ = 0.9795 Å, minimum of f′) and at a high-energy remote (λ = 0.9757 Å). 200 frames were recorded at each wavelength using an oscillation range of 0.5° and a crystal-to-detector distance of 320 mm.
The three selenium sites were located with SOLVE (Terwilliger & Berendzen, 1999 ) and were input into a maximum-likelihood heavy-atom parameter refinement using SHARP (de La Fortelle & Bricogne, 1997 ), which was followed by density modification with SOLOMON (Abrahams & Leslie, 1996 ) assuming a solvent content of 50%. A partial model was obtained with RESOLVE (Terwilliger, 2000 ) and was used for further manual building in the experimental electron-density map using TURBO-FRODO (Roussel & Cambillau, 1989 ).
The SRA domain of human UHRF1 was overexpressed in E. coli cells with typical yields of about 3 mg per litre of culture for the native protein and 1 mg per litre of culture for the SeMet-labelled protein. The SRA protein was purified by nickel-affinity chromatography with subsequent cleavage of the histidine tag by rTEV. Gel-filtration chromatography as a final purification step provided pure protein as judged by SDS–PAGE with Coomassie staining. Analysis by DLS experiments of the purified SRA protein in high-salt buffer showed a monodisperse solution (polydispersity of 9%) with a hydrodynamic radius of 2.7 nm and a calculated molecular weight of 34 kDa, corresponding to a monomeric species. A low reproducibility rate (<20%) of crystallization was achieved with the protein stored in high-salt buffer. On overnight dialysis of the protein against low-salt buffer, the crystallization rate increased to 70%. However, when the protein was purified in low-salt buffer at the gel-filtration step, the crystallization rate was high but the crystals dissolved within 2 d. SAXS experiments indicated monodispersity of the SRA solutions when the protein was in high-salt buffer or subsequently dialyzed against low-salt buffer but the occurrence of repulsive interactions when the protein was purified in low-salt buffer (Fig. 1 ). Numerous and stable protein crystals that were suitable for diffraction experiments were thus obtained with the SeMet-labelled protein purified in high-salt buffer and dialyzed against low-salt buffer.
The crystals belong to the trigonal space group P3221, with unit-cell parameters a = b = 53.78, c = 162.05 Å. They contain one molecule in the asymmetric unit, with a corresponding Matthews coefficient (Matthews, 1968 ) of 2.54 Å3 Da−1 and a solvent content of 52%. A MAD data set collected to 2.2 Å resolution allowed the structure determination of the human SRA domain. 73% of the atoms were built in the experimental electron-density map, attesting to the good quality of the MAD phases. A detailed description of the refined structure will be published elsewhere (PDB code 2pb7).
We thank the scientific staff of ESRF beamline ID14-EH4 (Grenoble, France) and DESY beamline X33 (Hamburg, Germany) for providing excellent data-collection facilities, Dino Moras for his contribution and discussions, the Structural Biology and Genomics Platform (especially Edouard Troesch) for cloning, Guillaume Bec for technical assistance with the Mosquito nanodrop robot and Hélène Nierengarten for mass-spectrometric analysis. This work was supported by the Region Alsace, the European Commission as SPINE2-Complexes (contract No. LSHG-CT-2006-031220) and ‘Cancéropôle Grand-Est’.