Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth disease

Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth Disease.

Abstract

Glycyl-tRNA synthetase (GlyRS) is one of a group of enzymes that catalyze the synthesis of aminoacyl-tRNAs for translation. Mutations of human and mouse GlyRSs are causally associated with Charcot–Marie–Tooth disease, the most common genetic disorder of the peripheral nervous system. As the first step towards a structure–function analysis of this disease, native human GlyRS was expressed, purified and crystallized. The crystal belonged to space group P4₃2₁2 or its enantiomorphic space group P4₁2₁2, with unit-cell parameters a = b = 91.74, c = 247.18 Å, and diffracted X-rays to 3.0 Å resolution. The asymmetric unit contained one GlyRS molecule and had a solvent content of 69%.

1. Introduction

Aminoacyl-tRNA synthetases (ARSs) are ancient enzymes presumptively involved in the establishment of the genetic code. They catalyze the first step of protein biosynthesis; that is, the ligation of each of the 20 amino acids onto the 3′ ends of their cognate tRNAs, which bear the specific trinucleotide anticodons of the code. Each amino acid has a specific synthetase, giving a total of 20 ARSs in most organisms. In general, the 20 ARSs are evenly separated into two classes according to their sequences and structural homology (Webster et al., 1984 ▶; Ludmerer & Schimmel, 1987 ▶; Eriani et al., 1990 ▶; Cusack et al., 1990 ▶).

Our understanding of the functions of ARSs in higher eukaryotes has greatly expanded over the past few years. Novel biological activities have been discovered for several ARSs and for auxiliary factors associated with ARSs (Wakasugi & Schimmel, 1999 ▶; Levine et al., 2003 ▶; Sampath et al., 2004 ▶; Yang et al., 2004 ▶; Park et al., 2005 ▶; Jordanova et al., 2003 ▶; Lee et al., 2006 ▶). Among others, the class II glycyl-tRNA synthetase (GlyRS) is connected with specific human diseases. Antibodies against GlyRS were detected in the sera of patients suffering from polymyositis or dermatomyositis and interstitial lung diseases (Freist et al., 1996 ▶). More significantly, mutations in both human and mouse GlyRSs are causally associated with one type of Charcot–Marie–Tooth disease. This inherited disorder of the peripheral nervous system (PNS) is the most common genetic disease of the PNS, affecting approximately 1 in 2500 people (Skre, 1974 ▶).

Human GlyRS, along with other eukaryotic and archeal GlyRSs, forms α₂ homodimers. In contrast, some bacterial GlyRSs (e.g. Escherichia coli) form α₂β₂ heterotetramers, while others (e.g. Thermus thermophilus) are eukaryote-like α₂ homodimers. The two solved structures of GlyRS are from bacterial sources: T. thermophilus GlyRS as an α₂ homodimer (Logan et al., 1995 ▶) and the α-chain of the α₂β₂ Thermotoga maritima GlyRS (PDB code 1j5w). Human GlyRS shares less than 30% sequence identity to T. thermophilus GlyRS, which contains an N-terminal characteristic class II catalytic domain (with three conserved sequence motifs built around a seven-strand mostly antiparallel β-sheet framework) and a C-terminal anticodon-recognition domain. Compared with T. thermophilus GlyRS, human GlyRS has extensions at both the N- and C-­termini and three insertions (of various sizes) into the catalytic domain (Fig. 1 ▶). To date, at least six different CMT-associated mutant alleles of the GlyRS gene (GARS) have been identified in the human population (Antonellis et al., 2003 ▶; Sivakumar et al., 2005 ▶; Del Bo et al., 2006 ▶) and one in the mouse (Seburn et al., 2006 ▶). The mutations are spread out over the structure of the homologous T. thermophilus GlyRS (Fig. 1 ▶). Except for D500N, which resides within the third insertion of the catalytic domain, all mutations are contained in the conserved regions shared by human and T. thermophilus GlyRS (Mazauric et al., 1998 ▶). Using the existing structural information on GlyRS, no explanation of the connection to CMT disease has been obtained.

Figure 1.

Mapping of the human and mouse CMT disease-causing mutations on the homologous T. thermophilus GlyRS structure. Compared with T. thermophilus GlyRS, human GlyRS has extensions at both the N- and C-termini and three insertions of various sizes into the catalytic domain. The three conserved sequence motifs in the catalytic domain are colored magenta, pink and orange, respectively.

Thus, in order to establish a structural framework for investigating the association of GlyRS with CMT disease, we sought to determine the crystal structure of human GlyRS and the GlyRSs encoded by the CMT mutant alleles. As the first step, we here describe the expression, purification, crystallization and preliminary X-ray diffraction analysis of native human GlyRS.

2. Protein expression and purification

The original human GlyRS gene GARS (accession No. BAA06338), including the mitochondrial-specific coding region, was cloned into a pET-21a(+) vector (EMD Biosciences, Inc., Novagen, Madison, WI, USA) by Anthony Antonellis at the National Human Genome Research Institute of the NIH. This clone was used as a template to create plasmid pCMM171, in which the mitochondrial-specific region (54 amino acids) was removed from the N-terminus. The new construct represents the cytosolic human GlyRS (685 amino acids) linked with a C-terminal coding sequence for a dipeptide spacer (Leu-Glu) immediately preceding a His₆ tag.

A 2 l culture of Luria–Bertani broth containing 100 mg ml⁻¹ ampicillin was inoculated with a 5 ml overnight culture of E. coli BL21(DE3)/pCMM171 and grown at 303 K to an OD₆₀₀ of 0.5–0.6. Human GlyRS expression was then induced by the addition of 1 mM isopropyl β-d-thiogalactopyranoside (IPTG). The E. coli cells were grown for a further 12 h before being harvested by centrifugation at 5000g for 20 min. The cell pellets were washed with a buffer containing 40 mM Tris–HCl pH 8.0, 250 mM NaCl and resuspended in 25 ml Ni–NTA buffer A (40 mM Tris–HCl pH 8.0, 250 mM NaCl, 10 mM imidazole, 1 mM PMSF). The cells were disrupted by three passes through a French press. The lysate was centrifuged at 20 000g for 50 min to remove cell debris. The supernatant was filtered through a 0.22 µm syringe filter and applied onto an Ni–NTA (Qiagen, Valencia, CA, USA) column previously equilibrated with Ni–NTA buffer A. The column was washed with 20 column volumes of washing buffer (40 mM Tris–HCl pH 8.0, 250 mM NaCl, 15 mM imidazole). Using an imidazole gradient, the protein was eluted from the column at approximately 100–210 mM imidazole. The GlyRS-containing fractions were pooled, concentrated and applied onto a MonoQ (HR 16/10) column (Amersham Bioscience, Piscataway, NJ, USA) equilibrated with MonoQ buffer A (20 mM Tris–HCl pH 7.8, 20 mM NaCl and 1 mM DTT). The column was eluted with buffer containing an NaCl gradient and the protein eluted at a concentration of 200–270 mM NaCl. Glycerol [25%(v/v) final concentration] was added to the eluted protein before it was flash-frozen and stored at 193 K. The protein remained fully active for aminoacylation after it was stored.

3. Crystallization

Human GlyRS was buffer-exchanged into 10 mM HEPES pH 7.0 with 20 mM NaCl before being concentrating to 8 mg ml⁻¹ using a Centricon centrifugal filter (Millipore, Billerica, MA, USA). High-throughput crystallization screens were set up with a Mosquito crystallization robot (TTP Labtech, Royston, England) using the sitting-drop vapor-diffusion method. The commercial screens Index (Hampton Research, Aliso Viejo, CA, USA) and Structure Screen (Molecular Dimensions Ltd, Apopka, FL, USA), as well as in-house PEG-based and ammonium sulfate-based screens, were used. 150 nl protein sample was mixed with an equal volume of reservoir solution in each well of 96-well plates at room temperature. Crystallization was monitored at regular intervals with the CrystalPro imaging system (TriTek, Sumerduck, VA, USA). Crystallization hits were obtained from the screens and subsequently optimized with the Opti-Salts suite (Qiagen, Valencia, CA, USA). After optimization, plate-like crystals (0.1 × 0.1 × 0.02 mm; Fig. 2 ▶) were obtained within 2 d by vapor diffusion of 2 µl hanging drops (1 µl protein and 1 µl reservoir solution) against 1 ml reservoir solution consisting of 10% PEG 6000, 0.1 M HEPES pH 6.5, 0.01 M Tris–HCl pH 8.5, 0.5 M NaCl and 0.1 M magnesium acetate at room temperature. Crystals were soaked for 3–5 min in a freshly made cryoprotective solution containing the components of the reservoir solution plus 20%(v/v) glycerol. The soaked crystals were mounted on a nylon loop and flash-frozen in liquid nitrogen.

Figure 2.

Crystals of human GlyRS. The dimensions of the largest crystal are approximately 0.1 × 0.1 × 0.02 mm.

4. Preliminary X-ray analysis

Using the Stanford Automated Mounting system (SAM) at beamline 11-1 of the Stanford Synchrotron Radiation Laboratory (SSRL), pre-frozen crystals were mounted onto the goniometer one by one to screen for diffraction quality. The best crystal diffracted to 3.0 Å resolution (Fig. 3 ▶). A complete data set was recorded with an ADSC Quantum-315 CCD detector at a wavelength of 0.979 Å. The crystal was exposed for 10 s and rotated through a 0.3° oscillation per frame at a crystal-to-detector distance of 320 mm. Data were processed with DENZO and scaled with SCALEPACK from the HKL-2000 package (Otwinowski & Minor, 1997 ▶). The crystal belonged to space group P4₃2₁2 or the enantiomorphic space group P4₁2₁2, with unit-cell parameters a = b = 91.74, c = 247.18 Å. The Matthews coefficient was calculated to be 3.32 ų Da⁻¹, indicating one molecule of GlyRS per asymmetric unit and a solvent content of 69.2%. Data-collection statistics are given in Table 1 ▶.

Figure 3.

A diffraction image of a human GlyRS crystal that diffracted to 3.0 Å resolution. The crystal was exposed to the X-ray beam (0.979 Å wavelength) for 10 s and rotated through a 0.3° oscillation at a crystal-to-detector distance of 320 mm.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

X-ray source	SSRL beamline 11-1
Wavelength (Å)	0.97945
Space group	P43212 or P41212
Unit-cell parameters (Å)	a = b = 91.74, c = 247.18
Resolution (Å)	50–3.0 (3.12–3.0)
Unique reflections	22076
Completeness (%)	96.9 (78.0)
Redundancy	4.8 (2.8)
Rmerge† (%)	8.9 (34.7)
〈I/σ(I)〉	13.2 (3.0)

^† R _merge = , where I is the intensity of an individual reflection and 〈I〉 is the average intensity over symmetry equivalents.

We attempted to solve the structure by molecular replacement. A number of starting models were used for the search. These included the T. thermophilus GlyRS structure, the backbone conformation of the same structure with all side chains replaced by alanines and various homology models of human GlyRS generated with Robetta (http://www.robetta.org). In addition, various programs (AMoRe, MOLREP and Phaser) from the CCP4 package (Collaborative Computational Project, Number 4, 1994 ▶) were used. These attempts have not yet produced a successful solution, presumably because of the low sequence identity between human GlyRS and the existing homologous structures. Currently, a de novo structure determination is being pursued using crystals of selenomethionine-labeled protein.