Crystallization and preliminary crystallographic analysis of nosiheptide-resistance methyltransferase from Streptomyces actuosus in complex with SAM

The expression, purification and crystallization of nosiheptide-resistance methyltransferase (NSR) from Streptomyces actuosus is described.

Abstract

Nosiheptide-resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur-containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging-drop vapour-diffusion method in reservoir solution consisting of 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet-labelled NSR crystals extended to 1.90, 1.95 and 2.25 Å resolution, respectively, using synchrotron radiation. All crystals belonged to space group P2₁, with approximate unit-cell parameters a = 64.6, b = 69.6, c = 64.9 Å, β = 117.8°.

1. Introduction

Bacterial resistance to antimicrobial agents is an increasingly important problem. Resistance to their own antibiotics has been observed in some drug-producing strains such as Streptomyces actuosus, S. antibioticus and S. glaucogriseus, which produce nosiheptide. Nosiheptide is a sulfur-containing peptide antibiotic with a molecular weight of 1222 Da that belongs to a family of thiazole antibiotics (Prangé et al., 1977 ▶; Endo & Yonehara, 1978 ▶). It has been used in veterinary medicine as a treatment against Gram-positive bacteria and has poor solubility (Cromwell et al., 1984 ▶). Other sulfur-containing peptide antibiotics include thiostrepton, siomycin, sporangiomycin etc. They are closely related in structure to nosiheptide and possess identical modes of action (Thompson & Cundliffe, 1980 ▶). Nosiheptide irreversibly binds to 50S subunits of ribosome and inhibits the hydrolysis of guanosine 5′-triphosphate (GTP) by elongation factor G (EF-G), thus inhibiting elongation (Wimberly et al., 1999 ▶; Harms et al., 2008 ▶). Since nosiheptide binds 23S ribosomal RNA (rRNA; Harms et al., 2008 ▶), self-protection is required for normal translation in its producer strain. S. actuosus immunizes itself with the 23S rRNA methyltransferase nosiheptide-resistance methyltransferase (NSR), which contributes to resistance against nosiheptide by methylation at 2′-OH of adenosine 1067 (Li et al., 1990 ▶). It has been reported that nucleotides 1051–1108 of 23S rRNA in Escherichia coli are the natural substrate of NSR (Bechthold & Floss, 1994 ▶). The sequence is composed of a terminal stem, 1067 stem-loop, 1082 hairpin and 1095 stem-loop (Gutell et al., 1994 ▶; Lentzen et al., 2003 ▶; Robinow & Kellenberger, 1994 ▶).

From amino-acid sequence analysis using Pfam (Finn et al., 2008 ▶), NSR is predicted to be composed of an N-terminal RNA-binding domain (NTD; residues 1–107) and a C-terminal catalytic domain (CTD; residues 108–274) which binds S-adenosyl l-methionine (SAM), a universal methyl donor for methylations mediated by methyltransferases (MTases; Roje, 2006 ▶). To date, five distinct classes of SAM-dependent MTases have been identified based on structural differences. NSR belongs to the class IV SPOUT-family MTases, which also contains the SpoU (TrmH including single domain or with the addition of ribosome large subunit L30 and L5 domains) and the TrmD [tRNA methyltransferase including single domain or with the addition of a TRP (tryptophan repressor) domain] subfamilies (Mosbacher et al., 2005 ▶).

RNA MTases have been identified as clinically significant resistance determinants to a number of antibiotics. For example, AviRb (avilamycin resistance factor b) from S. viridochromogenes mediates resistance to the oligosaccharide antibiotic avilamycin (Mosbacher et al., 2005 ▶), Erm is responsible for macrolide–lincosamide–streptogramin resistance (Farrow et al., 2000 ▶), 16S rRNA methyltransferase FmrO from Microspora olivasterospora is involved in fortimicin A resistance (Ohta & Hasegawa, 1993 ▶), RlmB from E. coli has been predicted to inhibit certain antibiotics (Michel et al., 2002 ▶) and TSR (thiostrepton-resistance methyltransferase) from S. azureus has been reported to be resistant to thiostrepton (Dunstan et al., 2009 ▶). The structures of several of these RNA methyltransferases have been reported. However, the structure of NSR and more importantly the molecular mechanism of NSR-mediated 23S rRNA methylation remain un­known.

Here, we report the expression, purification, crystallization and preliminary crystallographic studies of native NSR, of NSR in complex with SAM and of selenomethionine-derivative NSR.

2. Materials and methods

2.1. Protein expression and purification

The cDNA encoding full-length nsr (GenBank U75434.1) was amplified by PCR using genomic DNA from S. actuosus (a gift from Professor Alastair Murchie, Fudan University, People’s Republic of China) as the template and then inserted into the pETduet vector (Novagen) (using the DH5α strain) using EcoRI and XhoI restriction sites. The construct was verified by sequencing and the plasmid was transformed into E. coli strain BL21 (DE3) competent cells. The transformants were grown at 310 K to an OD₆₀₀ of 0.6 in Luria broth medium containing 100 mg l⁻¹ ampicillin and were induced by adding 0.1 mM isopropyl β-d-1-thiogalactopyranoside. NSR was expressed as a fusion protein with a His tag at the N-terminus. After a further 12–16 h incubation at 289 K, the cells were pelleted and resuspended in lysis buffer containing 25 mM Tris pH 8.0, 150 mM NaCl and 5 mM imidazole supplemented with DNAse and protease inhibitors. The cells were lysed on ice using a French press and the solution was clarified by centrifugation at 12 000 rev min⁻¹ for 25 min at 277 K. The supernatant was applied onto six Ni–NTA columns (1 ml resin per column; GE Healthcare) pre-equilibrated with lysis buffer. After washing with buffer containing 25 mM Tris pH 8.0, 150 mM NaCl and 20 mM imidazole, the fusion protein was digested on the column with TEV protease for 4 h at 277 K. The molecular weight of the digested protein was 29 547 Da including the additional Ser-Glu-Phe from the TEV cleavage site and EcoRI restriction site. The eluted protein was then loaded onto a Source 15Q anion-exchange column (GE Healthcare) and eluted with a linear gradient of 0–0.5 M NaCl at a flow rate of 10 ml min⁻¹. The peak fractions were collected and further purified by gel-filtration chromatography on a Superdex 200 column (GE Healthcare) with buffer containing 10 mM HEPES pH 7.5, 250 mM NaCl and 5 mM dithiothreitol (DTT). NSR-containing fractions were adjusted to 10 mg ml⁻¹ and used for crystallization.

Selenomethionine-derivative NSR protein was expressed using E. coli strain BL21 (DE3) cultured in M9 minimal medium supplemented with 100 mg l⁻¹ lysine, 100 mg l⁻¹ phenyl­alanine, 100 mg l⁻¹ threonine, 50 mg l⁻¹ isoleucine, 50 mg l⁻¹ leucine, 50 mg l⁻¹ valine and 25 mg l⁻¹ selenomethionine (Acros). Expression and purification procedures were performed as for wild-type NSR. Full incorporation of selenomethionine was verified by ESI mass spectrometry.

2.2. Protein crystallization

To obtain the NSR–SAM complex, NSR protein was incubated with SAM (New England Biolabs) on ice for 1 h with a 1:10 molar ratio of protein:SAM. Initial crystallization trials were performed using Crystal Screen, Index, SaltRX and PEG/Ion kits from Hampton Research and Wizard I and II kits from Emerald BioSystems at 293 K. These initial screens were set up using the hanging-drop vapour-diffusion method by mixing 1 µl protein solution and 1 µl reservoir solution. Initial conditions yielding crystals were further optimized by variation of the protein concentration, pH, precipitants and additives. We set up a total of approximately 600 conditions for optimization and screened three concentrations: 10, 6 and 4 mg ml⁻¹.

2.3. Data collection and processing

All crystals were mounted in nylon loops and flash-frozen in liquid nitrogen using reservoir buffer as cryoprotectant. Data collection was carried out on beamlines BL17A at Photon Factory, Japan and BL17U at SSRF, People’s Republic of China using crystals that had been flash-frozen at 100 K in a stream of cold nitrogen gas. A CCD detector was used. Data were indexed, integrated and scaled using the program HKL-2000 (Otwinowski & Minor, 1997 ▶). The refinement σ cutoff was set to 5 during data processing.

3. Results and discussion

Recombinant NSR protein was expressed, purified to homogeneity and used for crystallization (Figs. 1 ▶ a and 1 ▶ b). The purified NSR protein was estimated to be >98% pure (Fig. 1 ▶ c). The NSR protein precipitated above a concentration of 2 mg ml⁻¹ in gel-filtration buffer containing 25 mM Tris pH 8.0, 150 mM NaCl. We successfully improved the solubility of NSR in solution by increasing the NaCl concentration to 250 mM, which allowed NSR to be concentrated to >10 mg ml⁻¹ and used for crystallization trials. The crystals (Fig. 2 ▶) used for data collection were obtained by mixing 1 µl protein solution (4 mg ml⁻¹) with 1 µl reservoir solution containing 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. Large single cubic crystals appeared after 2 d and reached their maximum dimensions 6 d later. Crystals of NSR protein complexed with SAM and of the selenomethionine derivative of NSR were grown under similar conditions.

Figure 1.

NSR protein purification. (a) Ion-exchange (Source 15Q, GE Healthcare) profile of NSR protein. (b) Gel-filtration (Superdex 200, 10/300 GL, GE Healthcare) profile of NSR protein. Inset: one of the peak fractions was analyzed by SDS–PAGE with Coomassie Blue staining. Molecular weights are indicated in kDa.

Figure 2.

Crystals of selenomethionine-derivative NSR (a) and of NSR in complex with SAM (b) grown in 0.35 M ammonium chloride, 24%(w/v) PEG 3350, 0.1 M MES pH 5.7 at 293 K. The scale bar represents 0.1 mm.

In order to overcome any potential difficulties in using molecular replacement for structure determination, we produced crystals of selenomethionine-derivative NSR protein. Se-SAD (single-wavelength anomalous diffraction; Hendrickson, 1991 ▶) data were collected at a wavelength of 0.9788 Å using a single crystal. Data sets for the apo form of NSR, for NSR complexed with SAM and for SeMet-labelled NSR were also collected; the diffraction extended to 1.90, 1.95 and 2.25 Å resolution, respectively (Fig. 3 ▶). The crystals of SeMet-labelled NSR belonged to space group P2₁, with unit-cell parameters a = 64.6, b = 69.6, c = 64.9 Å, β = 117.8°. Two monomers are expected in the asymmetric unit, with a solvent content of about 41%. Data-collection statistics are summarized in Table 1 ▶. The structures were determined by the SAD phasing method and will be published elsewhere; molecular replacement was unsuccessful.

Figure 3.

X-ray diffraction pattern of the crystal of selenomethionine-derivative NSR. The ring indicates a resolution of 1.82 Å.

Table 1. Diffraction data statistics.

Values in parentheses are for the highest resolution shell.

	NSR	NSR–SAM	SeMet NSR
Wavelength (Å)	1.0055	0.9794	0.9788
Resolution (Å)	50.00–1.90 (1.97–1.90)	50.00–1.95 (2.02–1.95)	50.00–2.25 (2.33–2.25)
Space group	P21	P21	P21
Unit-cell parameters (Å, °)	a = 64.9, b = 69.2, c = 64.8, β = 117.8	a = 63.7, b = 69.2, c = 64.4, β = 117.6	a = 64.6, b = 69.6, c = 64.9, β = 117.8
Completeness (%)	97.4 (83.8)	99.2 (97.8)	99.5 (100.0)
Rmerge† (%)	5.8 (32.5)	5.3 (43.2)	9.1 (52.1)
I/σ(I)	33.3 (4.3)	31.7 (3.4)	20.4 (4.8)
Redundancy	7.1 (5.0)	7.2 (5.7)	7.6 (7.6)
Total No. of reflections	276005 (16845)	260071 (20229)	181982 (18134)
No. of unique reflections	38874 (3369)	36121 (3549)	23945 (2386)

^† R _merge = , where I _i(hkl) is the ith measurement of the intensity of reflection hkl and 〈I(hkl)〉 is the mean intensity of reflection hkl (Hall & McMahon, 2006 ▶).

The crystal structures of NSR and of NSR in complex with SAM may reveal the substrate-recognition and molecular mechanism of RNA methylation mediated by nosiheptide-resistance methyltrans­ferase. It will also expand our understanding of the mechanisms of antibiotic resistance.