crystallization papers
Crystallization and preliminary crystallographic studies of the D59A mutant of MicA, a YycF response-regulator homologue from Streptococcus pneumoniae
aIBLS, Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, Scotland, bLaboratoire de Genétique et Physiologie Bactérienne, E.A. 3036, Centre Hospitalo Universitaire de Rangueil, Université Paul Sabatier, 31403 Toulouse CEDEX, France, and cDepartment of Chemistry, University of Glasgow, Glasgow G12 8QQ, Scotland
*Correspondence e-mail: neil@chem.gla.ac.uk
RR02 (MicA) is an essential bacterial protein that belongs to the YycF family of response regulators and consists of two domains: an N-terminal receiver domain and a C-terminal effector domain. Streptococcus pneumoniae RR02 (MicA; residues 2–234) has been crystallized using the sitting-drop vapour-diffusion technique. The crystals belong to P21, with unit-cell parameters a = 46.46, b = 32.61, c = 63.35 Å, β = 90.01°. X-ray diffraction data have been collected to 1.93 Å resolution.
Keywords: S. pneumoniae; YycF homologue; response regulator; OmpR.
1. Introduction
Streptococcus pneumoniae is a bacterial pathogen responsible for diseases such as pneumonia, bacteraemia and meningitis and is a leading cause of illness and death in infants, the elderly and immunocompromised patients (AlonsoDeVelasco et al., 1995). Many strains of S. pneumoniae are becoming resistant to a wide range of antibiotics, including vancomycin and erythromycin, and new targets for antimicrobial agents are being sought.
Among such targets are the two-component signal-transduction systems, 13 of which exist in S. pneumoniae (Lange et al., 1999). The majority of two-component systems (TCS) consist of two distinct protein components: a histidine protein kinase (HPK) and a cytoplasmic response regulator (RR). The latter has two distinct domains: a receiver domain, which accepts the phosphoryl group from the HPK on an aspartic acid, and a DNA-binding domain, which controls gene expression (Stock et al., 1995). RR02 (MicA), a 26 kDa response-regulator protein, has been shown to be essential for bacterial growth. Knockout mutations in these genes lead to non-viable bacterial strains, thus making this a prime target for new antibacterial agent design (Brown et al., 2000; Lange et al., 1999; Throup et al., 2000). It has been shown that the two-component signal-transduction system HK02/RR02 is involved in competence repression under oxygen-limiting environments and was thus named Mic (microareobiosis control; Echenique & Trombe, 2001). It was proposed that the histidine kinase HK02 system contains a PAS domain, classically involved in redox reactions and in the protection of the cell against oxidative stress, in particular by repression of competence (Echenique et al., 2000). Under oxygen-limiting conditions, the HK02 histidine kinase sensor phosphorylates RR02 (MicA) on a highly conserved aspartate residue in the presence of magnesium ions, which in turn regulates the transcription of ComCDE (Echenique & Trombe, 2001) by an uncharacterized mechanism. The transfer of a phosphate group from HK02 to both the wild-type and mutant proteins was possible. However, mutation of Asp59 to Ala59 appears to destabilize the bond between Asp52 and phosphate: in the wild-type protein the half-life of RR02-PO4 was 20 min, whilst for the mutant (RR02-D59A-PO4) the half-life was 12 min (Echenique & Trombe, 2001).
In this paper, we report the cloning, purification, crystallization and preliminary crystallographic analysis of the RR02-D59A (MicA-D59A) mutant from S. pneumoniae.
2. Materials and methods
2.1. Protein expression and purification
The DNA coding for residues 2–234 of the mutated response regulator RR02 (MicA) was cloned into the pET33b vector (Novagen), which utilizes the T7lac promoter to drive recombinant protein overexpression (Echenique & Trombe, 2001). Expression plasmids were transformed into Escherichia coli BL21 (DE3) host strain (Stratagene) using a heat-shock technique. Several 1 l batches of LB-enriched growth media (Sigma) containing 100 µg ml−1 ampicillin were inoculated with 10 ml of an overnight culture. The cultures were shaken at 200 rev min−1 at 310 K until an OD600 of 0.5 was reached. IPTG (Melford) was then added to a final concentration of 1 mM in each culture to initiate overexpression of the RR02 (MicA) mutant. After induction, cells were allowed to grow for an additional 3 h at 310 K, harvested by spinning the cells at 5000g for 15 min at 277 K and resuspended in a buffer containing 25 mM Tris pH 7.5, 4 mM imidazole and 200 mM NaCl. Cells were lysed by sonication on ice (6 × 30 s) and centrifuged at 30 000g for 1 h to remove the insoluble fraction. The RR02 (MicA) mutant was purified by Ni-affinity The protein was loaded slowly (overnight at 277 K) onto a column previously equilibrated with buffer A (4 mM imidazole, 25 mM Tris, 200 mM NaCl pH 7.5) before elution at room temperature using a linear gradient of 0–100% buffer B (1 M imidazole, 25 mM Tris, 200 mM NaCl pH 7.5). The protein was further purified by gel filtration using a Superdex-75 column previously equilibrated in buffer C (25 mM Tris, 300 mM NaCl pH 7.5). Fractions containing the pure His-tagged protein were concentrated to between 5 and 10 mg ml−1 in an Amicon pressure cell before being used in crystallization trials.
2.2. Crystallization and data collection
Trials were set up using sitting-drop and hanging-drop vapour-diffusion techniques at both 277 and 293 K. The protein was mixed with the reservoir solution in a 1:1 ratio to produce 4 or 6 µl drops. Crystals unsuitable for diffraction were observed in a number of conditions after several days. A single crystal was observed after six months in condition No. 23 of the PEG/Ion screen; the reservoir contained 200 mM ammonium formate, 20%(w/v) PEG 3350 pH 6.6 (Fig. 1). Owing to the length of time taken to grow the initial crystal, no optimization was possible prior to data collection. The crystal was flash-cooled using dried paraffin oil as the cryoprotectant (Riboldi-Tunnicliffe & Hilgenfeld, 1999) and an almost complete data set was collected from a native crystal (Fig. 1), which diffracted to beyond 2.0 Å on beamline XRD-1 at ELETTRA, Trieste with a fixed wavelength of 1.0 Å. Data were collected at a detector distance of 140 mm from the crystal, with each frame being 1° in width. Owing to a fall in the intensity of the beam during data collection, the data were collected in three batches. Data were processed to a maximum resolution of 1.93 Å using MOSFLM (v.6.2.2; Leslie, 1992) and scaled using SCALA (Evans, 1993) and TRUNCATE (Collaborative Computational Project, Number 4, 1994). A summary of the crystallization conditions and X-ray data-collection statistics is given in Table 1.
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3. Results and discussion
Crystals of RR02 (MicA) measured 0.1 × 0.1 × 0.08 mm (Fig. 1) and diffracted to 1.93 Å resolution. With one monomer in the the determined Matthews coefficient is 1.94 Å3 Da−1 (Matthews, 1968) and the solvent content is 36.05%. A data set with a completeness of 98.7% and an Rsym of 0.087% was collected (full data-processing statistics can be found in Table 1).
As the β angle is very close to 90°, the data were processed in several space groups including the orthorhombic space groups P222, P2221, P21212, P212121, C222 and C2221 and the primitive monoclinic space groups P2 and P21. The merging statistics after SCALA (Evans, 1993) were much better in P21 than for the other space groups. Attempts are now under way to determine the structure by using as starting models RR02-Rec determined recently in our laboratory and the E. coli OmpR effector domain (Kondo et al., 1997; PDB code 1odd ).
Acknowledgements
We would like to thank the beamline staff at XRD1, ELETTRA for help and support during data collection. This work was supported by EC grant QLRK2-2000-00542 and a grant from the BBSRC.
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