|Home | About | Journals | Submit | Contact Us | Français|
A non-Pfam hypothetical protein SCO4226 of molecular weight 9 kDa from Streptomyces coelicolor A3(2) was overexpressed in Escherichia coli and the purified recombinant protein was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.0 Å resolution. The crystal belonged to space group P21, with unit-cell parameters a = 29.67, b = 67.00, c = 34.43 Å, α = γ = 90.00, β = 94.26°.
The Streptomyces coelicolor A3(2) open reading frame (ORF) SCO4226 encodes an 82-residue protein of unknown function with a theoretical molecular weight of 9049 Da (Redenbach et al., 1996 ). The results of searches using BLASTP and PSI-BLAST (http://www.ncbi.nlm.nih.gov; Altschul et al., 1997 ) suggested that SCO4226 is similar in primary sequence to the N-termini of three proteins: a guanylate cyclase (YP_001978230; 40% identity), an AraC-family member (YP_860235; 39% identity) and a putative two-component system (PhoR/PhoP system) regulator protein (ZP_01106696; 35% identity) (Sola-Landa et al., 2005 ; Fig. 1 ). To date, the structures of these proteins have not been reported.
Guanylate cyclases catalyze the cyclization of GTP and produce secondary messenger cGMP (Schulz et al., 2008 ; Schmidt et al., 2004 ). It has been proposed that the expression of soluble guanylate cyclase (sGC) might be alternated by increased superoxide production (Schulz et al., 2008 ). Lineage analysis of sGC suggests that sGC was originally an O2 sensor in primordial eukaryotes and then evolved to specifically recognize NO for more effective local cell-to-cell signalling (Fitzpatrick et al., 2006 ).
Most AraC-family members are transcription regulators and share a DNA-binding helix–turn–helix motif within the homologous C-terminus (Gallegos et al., 1993 ). However, the N-terminal regions of these regulators are highly variable and may be responsible for specific binding to different activator molecules (Gallegos et al., 1993 ; Martin & Rosner, 2001 ). One member of the AraC family, Escherichia coli SoxS, the counterpart of which in S. coelicolor is located downstream of SCO4226, has been reported to be involved in the regulation of responses to oxidative stress (Wu & Weiss, 1991 ; Li & Demple, 1994 ).
The PhoR/PhoP system, which lies upstream of SCO4226, also regulates the expression of oxidative stress-related proteins (Ghorbel et al., 2006 ; Sola-Landa et al., 2008 ). The membrane-bound protein PhoR senses the phosphate-limitation signal and undergoes self-phosphorylation. Phosphorylated PhoR then transfers the phosphate group to PhoP, resulting in the subsequent expression of more than 30 genes including Pho regulons (Sola-Landa et al., 2003 ). This system also plays an important role in the regulation of secondary metabolism and internal oxidative stress (Ghorbel et al., 2006 ). The primary sequence homology between SCO4226, the AraC-family member and the PhoR/PhoP system regulator protein implies a possible relation of their functions, which needs to be proved by structure-based functional interpretation and/or biochemical/biophysical investigations.
The ORF encoding SCO4226 was amplified by PCR using the cosmid DNA of S. coelicolor A3(2) as a template (provided by Dr Marie-Joelle Virolle at the Institute of Genetics and Microbiology, University Paris-Sud XI). Primers for amplification were designed based on the sequence available from http://streptomyces.org.uk (SCO4226, 2SCD46.40c). NdeI and XhoI restriction sites were incorporated into the sequences of the sense and antisense primers 5′-CCACATATGGCTCACTTCATGGACGTACAC-3′ and 5′-ATTCTCGAGCTATGCCGACAGGGGG-3′, respectively. After digestion with NdeI and XhoI restriction enzymes, the PCR product was cloned into the expression vector pET22b. E. coli Top10 cells were transformed with the ligation mixture. The sequence of the construct, which had no tags or extra residues, was confirmed by DNA sequencing. E. coli Rosetta (DE3) cells were transformed with plasmid pET22b-SCO4226 and grown on agar plates containing 50 µg ml−1 ampicillin. A single colony was verified and cultured in LB broth with 50 µg ml−1 ampicillin and 30 µg ml−1 chloramphenicol. Expression of the target protein was induced for 5 h at 310 K by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM when the OD600 reached 0.6. Cells were harvested by centrifugation at 7330g for 10 min and then resuspended in cold lysis buffer (20 mM Tris–HCl pH 8.0, 100 mM NaCl). The suspension was sonicated and clarified by centrifugation at 29 300g for 25 min at 277 K.
The high histidine-residue content of SCO4226 enabled us to purify the protein by affinity chromatography. The supernatant was loaded onto a nickel-chelating Sepharose Fast Flow column (Amersham Biosciences) pre-equilibrated with buffer containing 20 mM Tris–HCl pH 8.0, 200 mM NaCl. After the column had been washed with 20 ml of the same buffer, the binding protein was eluted by a gradual increase in imidazole concentration from 10 to 200 mM. The fraction that was eluted by binding buffer containing 200 mM imidazole was then further purified on a HiLoad 16/60 Superdex 75 column (Amersham Biosciences) equilibrated with buffer containing 20 mM Tris–HCl pH 8.0, 100 mM NaCl. The fractions containing the target protein were verified by SDS–PAGE.
Crystals of SCO4226 were obtained by the sitting-drop vapour-diffusion method at 289 K using screening kits from Hampton Research. Each sitting drop consisted of 1.0 µl 10 mg ml−1 protein solution (20 mM Tris–HCl pH 8.0, 100 mM NaCl) and 1 µl reservoir solution and was equilibrated against 100 µl reservoir solution. Crystals suitable for X-ray diffraction appeared within 3 d using a reservoir consisting of 1.6 M sodium citrate dehydrate pH 6.5. Crystals were flash-frozen in liquid nitrogen using a cryoprotectant consisting of the reservoir solution plus 25% glycerol. Diffraction data were collected to 2.00 Å resolution at 100 K using a Gemini R Ultra mounted with a 135 mm diagonal Ruby CCD detector and co-mounted Enhance (Mo) and Enhance Ultra (Cu) X-ray sources (Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences). The data were indexed and integrated with CrysAlisPro processing software (Oxford Diffraction) and scaled using SCALA (Evans, 1997 ) from the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ).
Despite lacking a His tag, SCO4226 showed a moderate affinity for the nickel-chelating Sepharose column. We therefore speculated that it may possess potential metal-binding ability. Atomic absorption spectra indicated that the purified protein contains a traceable amount of nickel, with an occupancy of 11%. Inspection of the primary sequences revealed nine histidine residues, three of which are well conserved among different species (Fig. 1 ). It is likely that the function of SCO4226 involves metal-ion ligation and the conserved histidine residues might be clustered in the tertiary structure in order to coordinate a metal ion.
Gel filtration indicated that SCO4226 eluted at a volume corresponding to about 20 kDa, which is approximately twice its theoretical molecular weight of 9 049 Da (Fig. 2 a). The purity was checked by SDS–PAGE (Fig. 2 b). Liquid-chromatography mass spectrometry (LTQ, Thermo Fisher) was used to further ensure that the collected fractions contained the target protein. A detected peptide (AGHKADEIHEVPLSA) matched the last 15 residues of SCO4226 exactly, whereas no matching fragment was detected in the E. coli protein database.
Plate-like crystals appeared in a drop consisting of 1.6 M sodium citrate dehydrate pH 6.5 in 3 d (Fig. 3 a). A set of diffraction data was collected from a single crystal to 2.00 Å resolution (Fig. 3 b). The crystal belonged to space group P21, with unit-cell parameters a = 29.67, b = 67.00, c = 34.43 Å, α = γ = 90.00, β = 94.26°. Based on the Matthews coefficient calculation, it was possible that each asymmetric unit contained one molecule, with a Matthews coefficient of 3.77 Å3 Da−1 and a solvent content of 67.39%, or two molecules, with a Matthews coefficient of 1.89 Å3 Da−1 and a solvent content of 34.8% (Matthews, 1968 ). However, the observation of a twofold noncrystallographic axis might better support the notion of two molecules per asymmetric unit. The data-collection statistics are summarized in Table 1 .
The gene cluster including SCO4226 was proposed to be involved in regulation of phosphate metabolism and/or response to oxidative stress. An increasing number of studies of these genes and their encoded proteins have been reported (Ghorbel et al., 2006 ; Sola-Landa et al., 2005 , 2008 ; Blanco et al., 2002 ), but the function of SCO4226 still remains unknown. The structure-based functional interpretation will provide us with new information about this protein.
This research was supported by grants 30628011 and 30470366 from the Chinese National Natural Science Foundation and projects 2006CB910202 and 2006CB806501 from the Ministry of Science and Technology of China.