Crystallographic analysis of a subcomplex of the transsulfursome with tRNA for Cys-tRNA^Cys synthesis

A subcomplex of the transsulfursome with tRNA^Cys (SepCysS–SepCysE–tRNA^Cys) represents the second reaction step of Cys-tRNA^Cys synthesis in an indirect pathway. Diffraction-quality crystals (2.6 Å resolution) of this complex were obtained. X-ray crystallographic analysis showed that the complex consists of a SepCysS dimer, a SepCysE dimer and one tRNA^Cys.

Abstract

In most organisms, Cys-tRNA^Cys is directly synthesized by cysteinyl-tRNA synthetase (CysRS). Many methanogenic archaea, however, use a two-step, indirect pathway to synthesize Cys-tRNA^Cys owing to a lack of CysRS and cysteine-biosynthesis systems. This reaction is catalyzed by O-phosphoseryl-tRNA synthetase (SepRS), Sep-tRNA:Cys-tRNA synthase (SepCysS) and SepRS/SepCysS pathway enhancer (SepCysE) as the transsulfursome, in which SepCysE connects both SepRS and SepCysS. On the transsulfursome, SepRS first ligates an O-phosphoserine to tRNA^Cys, and the mischarged intermediate Sep-tRNA^Cys is then transferred to SepCysS, where it is further modified to Cys-tRNA^Cys. In this study, a subcomplex of the transsulfursome with tRNA^Cys (SepCysS–SepCysE–tRNA^Cys), which is involved in the second reaction step of the indirect pathway, was constructed and then crystallized. The crystals diffracted X-rays to a resolution of 2.6 Å and belonged to space group P6₅22, with unit-cell parameters a = b = 107.2, c = 551.1 Å. The structure determined by molecular replacement showed that the complex consists of a SepCysS dimer, a SepCysE dimer and one tRNA^Cys in the asymmetric unit.

1. Introduction  

In the cell, each aminoacyl-tRNA synthetase specifically recognizes its assigned amino acid and cognate tRNA to form aminoacyl-tRNA, enabling accurate protein synthesis at the ribosome (Carter, 1993 ▸). In most organisms, cysteinyl-tRNA synthetase (CysRS) is responsible for Cys-tRNA^Cys synthesis by pairing a cysteine and its cognate tRNA^Cys; however, many methanogenic archaea do not have CysRS and cysteine-biosynthesis systems. These organisms use an indirect pathway in which two enzymes, O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS), are used for Cys-tRNA^Cys synthesis. Firstly, SepRS ligates O-phosphoserine to tRNA^Cys to form a mischarged intermediate, Sep-tRNA^Cys, which is then captured by SepCysS and converted to Cys-tRNA^Cys (Sauerwald et al., 2005 ▸). SepRS and SepCysS fail to form a stable binary complex, and the in vitro activities of both enzymes are very low (Zhang et al., 2008 ▸; Hauenstein et al., 2008 ▸; Liu et al., 2014 ▸), suggesting that other cofactors are necessary for this pathway. Recently, a third protein, SepRS/SepCysS pathway enhancer (SepCysE), was found to be essential for efficient SepRS and SepCysS activity by bridging the two enzymes to form a stable ternary complex. The three proteins constitute a functional unit in a 4:4:4 ratio with a molecular weight of approximately 540 kDa (Liu et al., 2014 ▸). Here, we call this functional unit the transsulfursome because of its function in two-step cysteine addition.

The previously determined crystal structure of SepRS in complex with tRNA^Cys revealed how SepRS recognizes the anticodon of tRNA^Cys to ensure the fidelity of the first reaction step (Fukunaga & Yokoyama, 2007 ▸). Little is known about the interaction between SepCysS and tRNA^Cys in the second reaction step. In addition, the previously reported structure of SepCysS in complex with the N-terminal domain of SepCysE [SepCysS–SepCysE(NTD)] elucidated the interaction between SepCysS and SepCysE (Liu et al., 2014 ▸), but the structure and function of the C-terminal domain of SepCysE [SepCysE(CTD)] remained unclear. How is tRNA^Cys bound to SepCysS during the second reaction step? Does SepCysS also show certain discriminants for Sep-tRNA^Cys to guarantee errorless reactions throughout the pathway? A structure of SepCysS–SepCysE with tRNA^Cys or Sep-tRNA^Cys is required to answer these questions. Here, we report the crystallo­graphic analysis of a subcomplex of the transsulfursome with tRNA^Cys (SepCysS–SepCysE–tRNA^Cys). This complex represents the second reaction step of tRNA^Cys synthesis in the indirect pathway involving the transsulfursome. The results of molecular replacement showed that the complex consists of two heterodimers of SepCysS–SepCysE and one tRNA^Cys in an asymmetric unit.

2. Materials and methods  

2.1. Macromolecule production  

Methods for the expression and purification of SepCysS–SepCysE were derived from previous research (Liu et al., 2014 ▸). Macromolecule-production information is summarized in Table 1 ▸. Two plasmids carrying Methanococcus jannaschii SepCysS and SepCysE, respectively, were co-transformed into Escherichia coli strain B834(DE3) plus pRARE2 (Novagen) by electroporation. The cells were grown in 3 l Luria broth medium at 310 K until OD₆₀₀ reached 0.6 and co-expression was then induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). After 16 h of cultivation at 298 K, the cells were harvested by centrifugation at 4500g for 30 min at 283 K and were resuspended in buffer A (50 mM HEPES–NaOH pH 8.0, 300 mM NaCl, 5 mM MgCl₂). The cells were disrupted by sonication in the presence of 0.5 mg ml⁻¹ lysozyme, 0.1 mg ml⁻¹ DNase I and a tablet of protease-inhibitor cocktail (cOmplete EDTA-free; Roche) followed by heat-treatment at 348 K for 30 min. The cell lysate was then centrifuged at 40 000g for 30 min at 298 K. The supernatant was applied onto a HisTrap HP column (GE Healthcare), and after washing out impurities with 20 mM imidazole the target protein complex was obtained by elution with a linear 20–500 mM imidazole gradient in buffer A. The collected fractions were then loaded onto a HiTrap Heparin HP column (GE Healthcare) to eliminate nucleic acids and were eluted by increasing the NaCl gradient to 1 M in buffer B (50 mM HEPES–NaOH pH 8.0, 5 mM MgCl₂). The eluted proteins were further purified using a HiLoad 16/60 Superdex 200 prep-grade column (GE Healthcare) equilibrated with buffer C (50 mM HEPES–NaOH pH 8.0, 300 mM NaCl, 5 mM MgCl₂, 10% glycerol). The purified protein was pooled, concentrated using an Amicon Ultra 30 kDa MWCO centrifugal concentrator (Millipore) and finally stored at 193 K.

Table 1. Macromolecule-production information.

Source organism	M. jannaschii
DNA source	M. jannaschii genome
SepCysS
Cloning site	NdeI–XhoI
Expression vector	pET-15b (Novagen)
Expression host	E. coli B834(DE3) plus pRARE2 (Novagen)
Complete amino-acid sequence of the construct produced†	MGSSHHHHHHSSGLVPRGSHNMELEGPYSKKFEVITLDINLDKYKNLTRSLTREFINLNPIQRGGILPKEAKKAVYEYWDGYSVCDYCHGRLDEVTCPPIKDFLEDIAKFLNMDCARPTHGAREGKFIVMHAICKEGDYVVLDKNAHYTSYVAAERAKLNVAEVGYEEEYPTYKINLEGYKEVIDNLEDKGKNVGLILLTHVDGEYGNLNDAKKVGKIAKEKGIPFLLNCAYTVGRMPVNGKEVKADFIVASGHKSMAASAPCGILAFSEEFSDKITKTSEKFPVKEIEMLGCTSRGLPIVTLMASFPHVVERVKKWDEELKKTRYVVDELEKIGFKQLGIKPKEHDLIKFETPVLDEIAKKDKRRGFFFYDELKKRGIGGIRAGVTKEIKMSVYGLEWEQVEYVVNAIKEIVESCK
SepCysE
Cloning site	NdeI–XhoI
Expression vector	Modified pCDF-Duet-1
Additional residues	Downstream box‡
Expression host	E. coli B834(DE3) plus pRARE2 (Novagen)
Complete amino-acid sequence of the construct produced†	MNHMRVEYSKDLIRKGISTISQLKKAKIRVEKDDKKISYKDAKPGKIDVNEFKKAIYLLIEADDFLYKKAPKHELNEEEAKEFCKLIIKCQEHLNKILANFGFEFEEKEIDEGALYIVSNKKLFKKLKNKNPNLKVVCTEGMLDIEDMRAIGVPEKALEGLKKKVEIARKNVERFIEKYKPEKIFVVVEDDKDELLYLRAKNLYNAEKLDADEILD
Synthesis of DNA template for tRNACys
Forward primer§	5′-TAATACGACTCACTATAGCCGGGGTAGTCTAGGGGCTAGGCAGCGGACT-3′
Middle primer	5′-CTAGGCAGCGGACTGCAGATCCGCCTTACGTGGGT-3′
Reverse primer¶	5′-TGGAGCCGGGGGTGGGATTTGAACCCACGTAAGGC-3′

^†Amino acids from the vector are underlined.

^‡The downstream box is a sequence element that enhances translation (Sprengart et al., 1996 ▸).

^§The T7 promoter is underlined.

^¶The underlined base G is modified by 2′-O-methylation.

M. jannaschii tRNA^Cys was transcribed in vitro as described previously, with modification (Nakamura et al., 2006 ▸). Firstly, PCR was performed to synthesize template DNA for tRNA^Cys transcription. The sequences of the DNA oligonucleotides used in PCR are listed in Table 1 ▸. After 5 h transcription at 310 K, the reaction was terminated and the product was purified by loading the sample onto 10% denaturing urea polyacrylamide gel electrophoresis (urea PAGE). The indicated tRNA band was excised under UV light and extracted from the gel using an Elutrap electroelution system (Whatman; Suzuki et al., 2015 ▸). The pooled tRNA^Cys was precipitated with ethanol and dissolved in a solution consisting of 20 mM HEPES–KOH pH 8.0, 5 mM MgCl₂; the purity was examined by urea PAGE prior to storage at 243 K.

2.2. Crystallization  

SepCysS–SepCysE was mixed with tRNA^Cys in different ratios and incubated at room temperature for 1 h. Crystallization drops were then set up with commercially available kits (The JCSG Core Suites I–IV, Qiagen) by mixing a 0.75 µl aliquot of the sample with an equivalent volume of reservoir solution. Using a sample consisting of SepCysS–SepCysE and tRNA^Cys mixed in a 1:1.2 ratio, with a final protein concentration of 16 mg ml⁻¹, initial crystals were obtained in several reservoir conditions as follows: (i) 0.1 M MES pH 6.0, 0.2 M Li₂SO₄, 35% MPD, (ii) 0.1 M Tris–HCl pH 7.0, 0.5 M ammonium sulfate, 10% glycerol, 30% PEG 600, (iii) 0.1 M MES pH 5.0, 2.4 M ammonium sulfate. After optimization of the buffer pH and precipitant concentration, high-quality crystals were obtained for data collection (Fig. 1 ▸). Crystallization information is summarized in Table 2 ▸.

Figure 1.

Crystals of SepCysS–SepCysE–tRNA^Cys (the crystals diffracted to 2.6 Å resolution).

Table 2. Crystallization.

Method	Sitting-drop vapour diffusion
Plate type	96-well
Temperature (K)	293
Protein concentration (mg ml−1)	16
Buffer composition of protein solution	20 mM HEPES–KOH pH 8.0, 100 mM KCl, 5 mM MgCl2, 5% glycerol
Composition of reservoir solution	0.1 M MES pH 5.3, 2.37 M ammonium sulfate
Volume and ratio of drop	0.75 µl, 1:1
Volume of reservoir (µl)	75

2.3. Data collection and processing  

For data collection, crystals of SepCysS–SepCysE–tRNA^Cys were soaked in cryoprotectant solution consisting of reservoir solution with a final concentration of 20% glycerol for several seconds before flash-cooling under a stream of nitrogen gas. The X-ray diffraction experiment was performed on beamline BL41XU at SPring-8, Harima, Japan (proposal Nos. 2014B1033 and 2015B1024). Diffraction data were collected with 0.01° oscillation and 0.01 s exposure time per image, with a total rotation of 180°. The data were processed using the CCP4 and XDS packages (Winn et al., 2011 ▸; Kabsch, 2010 ▸), and statistical information is summarized in Table 3 ▸.

Table 3. Data collection and processing.

Values in parentheses are for the outer shell.

Diffraction source	BL41XU, SPring-8
Wavelength (Å)	0.979
Temperature (K)	100
Detector	PILATUS3 6M
Crystal-to-detector distance (mm)	500
Rotation range per image (°)	0.01
Total rotation range (°)	180
Exposure time per image (s)	0.01
Space group	P6522
a, b, c (Å)	107.2, 107.2, 551.1
α, β, γ (°)	90, 90, 120
Mosaicity (°)	0.043
Resolution range (Å)	50–2.60 (2.75–2.60)
Total No. of reflections	448086 (72295)
No. of unique reflections	59262 (9258)
Completeness (%)	99.5 (98.6)
Multiplicity	7.56 (7.81)
〈I/σ(I)〉	15.97 (2.01)
R meas † (%)	9.7 (85.7)
Overall B factor from Wilson plot (Å2)	64.2

^† R _meas = , where 〈I(hkl)〉 and N(hkl) are the mean intensity of a set of equivalent reflections and the multiplicity, respectively.

3. Results and discussion  

The binding affinity of SepCysS–SepCysE to tRNA^Cys was investigated using an electrophoretic mobility shift assay (EMSA). The results showed that in solution, SepCysS–SepCysE bound to tRNA^Cys in a 2:1 ratio (Fig. 2 ▸ a), although the crystals were obtained at a 1:1.2 ratio of SepCysS–SepCysE to tRNA^Cys.

Figure 2.

Analysis of SepCysS–SepCysE–tRNA^Cys. (a) tRNA^Cys-binding analysis of SepCysS–SepCysE by EMSA. The molar ratios of SepCysS–SepCyE to tRNA^Cys in the samples in lanes 1, 2, 3 and 4 are 0:1, 1:1, 2:1 and 4:1, respectively. (b) SDS–PAGE analysis of crystals. A dissolved crystal was analyzed on a 15% SDS–PAGE gel stained with Coomassie Brilliant Blue. Purified SepCysS–SepCysE was used as a control. (c) The presence of tRNA^Cys in the crystals was confirmed by 10% urea PAGE stained with ethidium bromide. In vitro-transcribed tRNA^Cys was used as a control.

After obtaining crystals of SepCysS–SepCysE–tRNA^Cys, we checked the components of the crystals using SDS–PAGE and urea PAGE. Bands corresponding to the size of SepCysS and SepCysE in SDS–PAGE and of tRNA^Cys in urea PAGE were observed (Figs. 2 ▸ b and 2 ▸ c). SDS–PAGE showed degradation of SepCysS and SepCysE (Fig. 2 ▸ b). To clarify which parts of SepCysS and SepCysE were likely to degrade, we analyzed degraded SepCysS and SepCysE by N-terminal sequencing. The results showed that SepCysS and SepCysE degraded from the 16th and 18th residues at the N-terminus, respectively, which is consistent with the published structure of SepCysS–SepCysE(NTD), which has invisible N-termini for SepCysS and SepCysE. The dissolved crystals were further analyzed by TOF mass spectrometry, which confirmed the existence of SepCysS, SepCysE and tRNA^Cys with the corresponding sizes.

The diffraction pattern of snapshot images showed that the unit cell is very large, and the subsequent analysis of reflections with iMosflm (Battye et al., 2011 ▸) indicated that the length of one axis is over 500 Å. Thus, 0.01° oscillations were used to collect the data set in order to avoid reflection overlap (Fig. 3 ▸). The space group and unit cell were first estimated using POINTLESS from the CCP4 package (Evans, 2006 ▸). Based on the results from POINTLESS, diffraction data were successfully indexed, integrated, scaled and merged to a resolution of 2.6 Å using the XDS package. The space group of the SepCysS–SepCysE–tRNA^Cys crystals was defined as P6₁22 or P6₅22, with unit-cell parameters a = b = 107.2, c = 551.1 Å.

Figure 3.

X-ray diffraction image of a SepCysS–SepCysE–tRNA^Cys crystal. The resolution limit of 2.6 Å is marked with a dashed circle.

The structure of SepCysS–SepCysE–tRNA^Cys was resolved by molecular replacement using AutoMR from the PHENIX package (Adams et al., 2010 ▸; McCoy et al., 2007 ▸). The structures of SepCysS–SepCysE(NTD) from M. jannaschii (PDB entry 3wkr; Liu et al., 2014 ▸) and of tRNA^Cys from E. coli (PDB entry 1b23; Nissen et al., 1999 ▸) were used as search models. The SepCysS–SepCysE(NTD) and tRNA^Cys solutions were successfully found with a TFZ score of 93.4 in space group P6₅22. As a result, two heterodimers of a SepCysS dimer, a SepCysE dimer and one tRNA^Cys were observed in the asymmetric unit (Matthews coefficient V _M of 3.19 ų Da⁻¹ with 61% solvent content) in accordance with the binding ratio determined by EMSA. Model building of the missing C-terminal domain of SepCysE and refinement of the complex structure are currently being performed.