crystallization communications Acta Crystallographica Section F
Structural Biology Communications ISSN 2053-230X
Nipa Chongdar,a Saumya Dasgupta,a Ajit Bikram Dattab* and Gautam Basua* a
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India, and bDepartment of Biochemistry, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India
Correspondence e-mail: [email protected], [email protected]
Received 12 March 2014 Accepted 9 May 2014
Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase from Escherichia coli The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln. To properly understand this distinctive GluRS–tRNA interaction it is important to pursue detailed structure–function studies; however, because of the fact that tRNA– GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure–function correlation studies must also be phylumspecific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNAGlu–GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported.
1. Introduction
# 2014 International Union of Crystallography All rights reserved
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The core function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNAGlu (Kern et al., 1979; Breton et al., 1986), but not all bacterial GluRS restrict their tRNA-glutamylation to only the cognate tRNA (tRNAGlu) (Lapointe et al., 1986; Lamour et al., 1994; Dasgupta & Basu, 2014). In bacteria such as Escherichia coli and Thermus thermophilus that possess glutaminyl-tRNA synthetase (GlnRS), the cognate aminoacylating enzyme for tRNAGln, GluRS exclusively glutamylates tRNAGlu (Kern et al., 1979; Dasgupta et al., 2009; Becker & Kern, 1998). On the other hand, in bacteria that lack GlnRS, such as Bacillus subtilis, Thermosynechococcus elongatus and Mycobacterium tuberculosis, GluRS is equally capable of glutamylating the noncognate tRNAGln (Lapointe et al., 1986; Schulze et al., 2006; Paravisi et al., 2009). In addition, some bacteria, such as Thermotoga maritima, contain two copies of GluRS (GluRS1 and GluRS2) with different specificities for tRNAGlu and tRNAGln (Ito et al., 2010). The diverse tRNAGlx specificities of bacterial GluRS call for structure–function correlation studies that will yield a clear understanding of the mechanisms that control the GluRS–tRNAGlx interaction in bacteria. Because the precise nature of the GluRS– tRNAGlx interaction in bacteria is phylum-specific (Dasgupta et al., 2012; Dasgupta & Basu, 2014), biochemical work performed on the GluRS–tRNAGlx interaction in a bacterium from one phylum may not be compatible with the GluRS structure from a bacterium belonging to a distant phylum. Escherichia coli is a model bacterium, some strains of which are pathogenic. Most biochemical studies have been performed on E. coli GluRS (Eco-GluRS), both in our laboratory (Dasgupta et al., 2009, 2012; Saha et al., 2009, 2012) and in those of others (Sekine et al., 1996; Banerjee et al., 2004; Dubois et al., 2009). Recent reports have demonstrated how Eco-GluRS plays a role in pathogenesis by Acta Cryst. (2014). F70, 922–927
crystallization communications
Figure 1 (a) Homology-modelled structure of Eco-GluRS with colour-annotated domains (1–5). Disordered parts (in 4g6z and annotated as ‘mobile’), the position of Zn and the two surface residues that were mutated in this work (Lys236 and Glu328) are annotated. The Eco-GluRS model was built with superposed multiple templates (PDB entries 2cfo, 4gri and 4g6z) using MODELLER (Eswar et al., 2006). The percentage sequence identities (with Eco-GluRS) are 40.76% (2cfo), 39.49% (4g6z) and 35.45% (4gri), as calculated by ignoring disordered/invisible residues of the templates (residues 103–121 and 379–443 in 4g6z; residues 398–404 and 441–443 in 4gri). (b) Structural alignment of bacterial GluRS using MATRAS (Kawabata, 2003) with DSSP-assigned consensus secondary structure on the top row and residue conservation on the bottom row (colour coded according to the domains in Fig. 1a). Disordered regions (in 4g6z and 4gri) are highlighted in yellow and the corresponding residues are shown in grey. In addition, the proteobacteria-specific Arg266 (Eco-GluRS), the Cys residues in the zinc-binding motif and Lys236/Glu328 are highlighted with red, magenta and blue colours, respectively. The GluRS sequences are denoted by their four-character PDB codes (ECO stands for Eco-GluRS). Two crystal contacts in Bth-GluRS (4g6z), Glu242–Lys243 (c) and Ala334–Ala334 (d), leading to the two surface mutations K236E and E328A in Eco-GluRS.
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crystallization communications inducing multidrug tolerance upon binding to a eukaryote-like serine-threonine kinase (HipA; Germain et al., 2013; Kaspy et al., 2013). Crystal structures of GluRS from all of the species mentioned above, except for B. subtilis and E. coli, are available. The PDB entries are 1j09 (T. thermophilus; Nureki et al., 1995), 2cfo (T. elongatus; Schulze et al., 2006), 2ja2 (M. tuberculosis), 2o5r (T. maritima GluRS1) and 3afh (T. maritima GluRS2; Ito et al., 2010). In addition,
crystal structures of GluRS from Burkholderia thailandensis (PDB entry 4g6z; Baugh et al., 2013) and Borrelia burgdorferi (PDB entry 4gri; T. E. Edwards and M. C. Clifton, unpublished work) are also available. Based on the occurrences of GlnRS in their genomes (Dasgupta & Basu, 2014), the GluRS in B. thailandensis (a GlnRScontaining bacterium) and B. burgdorferi (a GlnRS-lacking bacterium) are expected to be tRNAGln-discriminatory and tRNAGlnnondiscriminatory, respectively. Figs. 1(a) and 1(b) show a homology model for Eco-GluRS (with PDB entries 2cfo, 4g6z and 4gri as templates), its various domains and a structural alignment of all known bacterial GluRS structures and Eco-GluRS. The homology model of Eco-GluRS is not precise enough to reveal the subtleties of GluRS–tRNAGlu interaction in E. coli owing to the absence of some specific features of Eco-GluRS in the template structures. For example, the proteobacterial-specific residue Arg266, which is present in a tRNA-interacting loop of Eco-GluRS (Dasgupta et al., 2012), is absent in all known bacterial GluRS structures except for that of B. thailandensis GluRS (Bth-GluRS; see Fig. 1b). The structure of Bth-GluRS (Fig. 1b), however, is unsuitable for detailed structural studies on Eco-GluRS not only because the SWIM-like domain of Bth-GluRS (100C-x-M-xn-W-x-P129) lacks the characteristic ‘C-x-C-xn-C-x-H’ zinc-binding motif of Eco-GluRS (Banerjee et al., 2004), but also because a long stretch of residues (103–121) encompassing the zinc-binding motif are disordered in the crystal structure 4g6z. Similarly, residues 379–443 in the functionally important Cterminal domain (domain 5) are also disordered in 4g6z. In order to overcome these shortcomings, it is imperative that the structure of Eco-GluRS be determined. Here, we report the expression, purification and preliminary crystallization screening of an engineered version of Eco-GluRS in which two surface residues, Lys236 and Glu328, were mutated to facilitate crystal packing.
2. Methods 2.1. Design of surface-engineered Eco-GluRS
Figure 2 (a) SDS-PAGE [8%(w/v)] analysis of dd-GluRS: lane 1, (6His)-(SUMO2 tag)(Gly)-(dd-GluRS) (64.7 kDa); lane 2, (Gly)-(dd-GluRS) (53.8 kDa) and 6HisSUMO2 (10.9 kDa) after SENP2 digestion; lane 3, purified fraction of (Gly)-(ddGluRS); lane 4, molecular-weight markers (labelled in kDa). (b) CD spectra of 5 mM wild-type Eco-GluRS and dd-GluRS in 50 mM pH 7.5 phosphate buffer containing 100 mM KCl at 298 K. (c) The time course of product (Glu–tRNAGlu) formation during glutamylation of Eco-tRNAGlu by wild-type Eco-GluRS and ddGluRS.
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Eco-GluRS is known to resist crystallization (Vanoni et al., 2005). Previous attempts to crystallize Eco-GluRS in our laboratory were also unsuccessful. A number of factors might have contributed to this failure, including inter-domain dynamics, but this can be ruled out for Eco-GluRS since quite a few homologous GluRSs from other bacteria have successfully been crystallized. Another factor responsible for the observed resistance of Eco-GluRS crystallization could be unfavourable crystal contacts. In order to identify a subset of EcoGluRS residues that might be hindering lattice formation, we compiled a list of residue pairs involved in pairwise crystal contacts (pccs) in an evolutionarily close homologue (Bth-GluRS; PDB entry 4g6z). Using a multiple sequence alignment of bacterial GluRS, we pruned the pccs by removing (i) residues that were identical between Eco-GluRS and Bth-GluRS and (ii) residues that were mostly conserved in the multiple sequence alignment. Physical interactions between all the remaining residue pairs in the pruned pccs were visually inspected in the Bth-GluRS structure and compared with the Eco-GluRS model (built with 4g6z as the template). This allowed us to identify two energetically favourable pairs in 4g6z that were unfavourable in Eco-GluRS. The first pair involves residues Glu242 and Lys243 in 4g6z, which form an electrostatically favourable salt bridge (Fig. 1c), but the interaction between corresponding residues in Eco-GluRS (Lys236–Lys237) is electrostatically unfavourable (Fig. 1c). The second residue pair Ala334–Ala334 in 4g6z (Fig. 1d) is electrostatically neutral whereas the corresponding pair in EcoGluRS (Glu328–Glu328) (Fig. 1b) is electrostatically unfavourable. Acta Cryst. (2014). F70, 922–927
crystallization communications In order to remove these two plausible unfavourable crystal contacts, we introduced two mutations (K236E/E328A) in Eco-GluRS and attempted crystallizing the double mutant (dd-GluRS).
mutations were confirmed by DNA sequencing (Applied Biosystems).
2.2. Cloning of Eco-GluRS in pETSUMO2
2.3. Overexpression and purification
The GluRS-encoding gene of E. coli was amplified from plasmid DNA PLQ7619 using appropriate primers. The amplified product was cloned in a pETSUMO2 vector (Datta & Wolberger, unpublished work) using an In-Fusion PCR Cloning Kit (Clontech). The resulting plasmid coded for Eco-GluRS with an N-terminal 6His-SUMO2 tag and a Gly residue [(6His)-(SUMO2 tag)-(Gly)-(Eco-GluRS)]. K236E and E328A mutations were introduced into Eco-GluRS by PCR-based site-directed mutagenesis using appropriate primers. The
Cultures of E. coli Rosetta2 (DE3) strain transformed with the recombinant plasmid were grown in Circlegrow (MP Biomedicals) medium supplemented with 100 mg ml1 ampicillin at 310 K until the OD600 reached 1; protein expression was induced with 0.5 mM isopropyl -d-1-thiogalactopyranoside (IPTG) for 16 h at 288 K. The cells were harvested by centrifugation (5000g for 10 min) followed by resuspension in ice-cold lysis buffer (20 mM sodium phosphate pH 8, 500 mM NaCl, 5 mM MgCl2, 10 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and DNase I) and lysed in a microfluidizer (Microfluidics) at 277 K. The crude lysate was clarified by centrifugation (20 000g for 45 min at 277 K) followed by filtration through a 0.22 mm membrane (Millipore). The recombinant GluRS was purified from the clarified lysate using a 5 ml HisTrap HP affinity column (GE Healthcare) in 20 mM phosphate buffer pH 8 containing 400 mM NaCl, 20 mM ZnCl2 under a linear gradient of imidazole (20– 400 mM). The purified fusion protein [(6His)-(SUMO2 tag)-(Gly)(Eco-GluRS)] was dialyzed in 20 mM sodium phosphate buffer pH 8 containing 150 mM NaCl, 20 mM ZnCl2, 10 mM -mercaptoethanol for 16 h at 277 K, during which the 6His-SUMO2 tag was cleaved by adding SENP2 protease (3 mg SENP2 was added to 150 mg fusion protein; lanes 1 and 2 of Fig. 2a). The mixture was passed through another HisTrap HP affinity column (GE Healthcare) to remove the (6His)-(SUMO2) tag and SENP2. The flowthrough, containing the untagged GluRS, was estimated to be of >95% purity as judged by SDS–PAGE followed by Coomassie staining (lane 3 of Fig. 2a). It should be noted that in the absence of the additional glycine between the 6His-SUMO2 tag and the Met1 of Eco-GluRS, cleavage efficiency was substantially reduced (40–50%) under identical conditions, even with a higher SENP2:protein ratio [increased to 0.1:1(w:w) from 0.02:1(w:w)]. All the chromatographic steps were performed ¨ KTA Avant liquid-chromatography system (GE Healthusing the A care). Purified (Gly)-(Eco-GluRS) was dialyzed in storage buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM -mercaptoethanol, 50 mM ZnCl2), concentrated to 35 mg ml1 using centrifugal concentrators of molecular-weight cutoff 30 kDa (Vivaspin 20, Sartorius AG) and stored at 193 K after flash-freezing in liquid nitrogen.
2.4. Secondary-structure analysis and glutamylation assay
Figure 3 (a) A crystal of dd-GluRS obtained using the hanging-drop vapour-diffusion method (see x2). (b) A typical diffraction pattern of the GluRS crystal (oscillation ˚ from the centre). image) annotated with resolution arcs (8, 5, 4 and 3 A
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Changes in the secondary-structure contents of the wild-type and mutant Eco-GluRS were assessed from circular-dichroism (CD) spectra (200–260 nm) of 5 mM protein (Fig. 2b) recorded in a J-815 CD spectropolarimeter (Jasco) with a 2 mm cuvette in 50 mM phosphate buffer pH 7.5 containing 100 mM KCl at 298 K. The enzymatic activities of wild-type Eco-GluRS and dd-GluRS were determined by in vitro aminoacylation assays where the rate of product ([3H]-l-Glu–tRNAGlu) formation was monitored as a function of time. The reactions were carried out with 5 mM E. coli tRNAGlu in 50 mM HEPES pH 7.5, 0.1 mM unlabelled l-Glu, 16 mM MgCl2, 2 mM ATP, 0.8 mM -mercaptoethanol and [3H]-l-Glu (0.5 ml of stock per 100 ml of assay mixture) at 310 K as described previously (Dasgupta et al., 2009). Protein concentrations used per assay point were 5.4 nM. Chongdar et al.
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crystallization communications Table 1 Data collection and processing statistics. Values in parentheses are for the outermost shell. Diffraction source ˚) Wavelength (A Temperature (K) Exposure time (s) Space group ˚ , ) Unit-cell parameters (A
RU 300 [Blue optics] 1.54178 110 300 C2 a = 213.05, b = 61.52, c = 102.74, = 96.25 29.89–3.30 (3.42–3.30) 67555 20231 99.8 (100) 3.34 (3.38) 8.8 (1.5) 2.19 6.2 (49.0) 3.17 2
˚) Resolution range (A Observed reflections Unique reflections Completeness (%) Multiplicity hI/(I)i Average mosaicity ( ) Rmerge† (%) ˚ 3 Da1) VM (A Molecules per asymmetric unit † Rmerge =
P
hkl
P
i
jIi ðhklÞ hIðhklÞij=
P
hkl
P
i Ii ðhklÞ.
2.5. Crystallization and X-ray diffraction data collection
Initial crystallization screenings were performed using commercially available screens (Index and PEGRx; Hampton Research) and Morpheus (Gorrec, 2009) at 293 K in 24-well XRL plates (Molecular Dimensions) using the hanging-drop vapour-diffusion method. Morpheus condition 90 [0.1 M MOPS/HEPES–Na pH 7.5, 0.02 M each of l-Glu.Na, dl-Ala, dl-Lys.HCl, Gly and dl-Ser, 10%(w/v) PEG 8000, 20%(v/v) ethylene glycol] yielded crystal clusters. The crystallization condition was further optimized by varying the pH (7.3–7.9), the concentrations of PEG 8000 [10–14%(w/v)] and ethylene glycol [14–28%(v/v)] and the protein:crystallization cocktail ratio [1:1–1:2(v:v)]. Single crystals suitable for diffraction (Fig. 3a) were obtained from drops prepared with 1 ml 20 mg ml1 dd-GluRS and 1.2 ml of the optimized crystallization cocktail [0.1 M MOPS/ HEPES–Na pH 7.7, 0.02 M each of l-Glu.Na, dl-Ala, dl-Lys.HCl, Gly and dl-Ser, 14%(w/v) PEG 8000, 22%(v/v) ethylene glycol]. Crystals grew to a size of about 300 100 50 mm in two weeks and ˚ (Fig. 3b). typically diffracted to a resolution of 3.5 A Prior to X-ray diffraction data collection, the crystals were soaked in mother liquor containing an increasing concentration of ethylene glycol, from 22% to 32%, in 2% steps with about 1 min of soaking at each step. A few initial images were collected and processed with d*TREK (Pflugrath, 1999) to identify the space group and unit-cell parameters. Final X-ray diffraction data were collected in-house at 110 K in steps of 1 over 170 total rotation using a Rigaku RU300 generator with a Cu anode and an R-AXIS IV++ image-plate detector. The data were integrated, scaled and merged using d*TREK. The crystals belonged to the monoclinic space group C2, ˚, = with unit-cell parameters a = 213.05, b = 61.52, c = 102.74 A 96.25 . The data statistics are summarized in Table 1. The merged intensity data were imported into the CCP4 suite (Winn et al., 2011) and converted to structure-factor amplitudes using CTRUNCATE (French & Wilson, 1978). The solvent content was estimated from the Matthews coefficient (Matthews, 1968) using a molecular mass of 53.8 kDa. Molecular-replacement phasing was attempted with either Phaser (McCoy et al., 2007) or MOLREP (Vagin & Teplyakov, 2010).
3. Results and discussion Based on the crystal packing of Bth-GluRS structure (PDB entry 4g6z), the designed double mutant (K236E/E328A) of Eco-GluRS (dd-GluRS) was successfully crystallized. The corresponding wild-
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type GluRS failed to crystallize under similar conditions. The secondary structure contents of dd-GluRS and the wild-type protein were indistinguishable, as judged from CD studies (Fig. 2b). The enzymatic activities (rate of formation of the product Glu–tRNAGlu) of wild-type Eco-GluRS and dd-GluRS were also very similar as indicated by the slopes of product formation at early time points (Fig. 2c). Therefore, not only did the engineered GluRS crystallize but the mutations that enabled crystallization of the protein did not affect its overall structure or the function. The volume of the asymmetric unit in the dd-GluRS crystal was compatible with the presence of either two or three monomers in the asymmetric unit, with a volume per unit molecular weight of the ˚ 3 Da1 and a calculated solvent content protein (VM) of 3.02–2.03 A of 59.65–39.48% (Matthews, 1968). Molecular-replacement trials performed using either PDB entry 2cfo (GluRS from T. elongatus; Schulze et al., 2006) or 4g6z (B. thailandensis GluRS; Baugh et al., 2013) as search models consistently showed two monomers in the asymmetric unit. However, neither of the search models yielded electron-density maps that were good enough to permit further building and refinement of the model with reasonable R factors. The two domains of GluRS (see Fig. 1) show rigid-body dynamics in superimposed structures of homologous GluRS. To address this issue, we also tried molecular replacement by defining two domains from PDB entry 2cfo (2–384 and 385–485) as separate ensembles in a single search. Although this improved the map quality, it was still not possible to complete the structure. The poor molecular-replacement phasing could be due to low resolution and/or poor data quality. To circumvent this problem, we are currently pursuing further optimizations to obtain higher resolution data with options of including experimental phasing techniques. Efforts to crystallize Eco-GluRS bound to ligands (tRNAGlu and ATP) are also under way. In summary, we have successfully crystallized Eco-GluRS by introducing two point mutations. dd-GluRS crystallized in space group C2, different from the space group (P41212) of the template (4g6z) used for optimizing the crystal contacts in Eco-GluRS. Nonetheless, the fact that the engineered crystal contacts played a role in successful crystallization can certainly be concluded from the fact that the wildtype protein did not crystallize under identical conditions. The mutated residues therefore either play a role in crystal nucleation or stabilize the final crystal lattice. The latter can only be confirmed once the structure has been solved, although all preliminary molecularreplacement solutions point towards crystal contacts by K236E and E328A. GB acknowledges financial supported by a Wellcome Fellowship. We thank Adaitya SENP2 protease and Maura carefully.
support from CSIR, India. ABD is Trust–DBT Alliance Intermediate Behera for his help in preparing the Hurley for reading the manuscript
References Banerjee, R., Dubois, D. Y., Gauthier, J., Lin, S.-X., Roy, S. & Lapointe, J. (2004). Eur. J. Biochem. 271, 724–733. Baugh, L. et al. (2013). PLoS One, 8, e53851. Becker, H. D. & Kern, D. (1998). Proc. Natl Acad. Sci. USA, 95, 12832–12837. Breton, R., Sanfac¸on, H., Papayannopoulos, I., Biemann, K. & Lapointe, J. (1986). J. Biol. Chem. 261, 10610–10617. Dasgupta, S. & Basu, G. (2014). BMC Evol. Biol. 14, 26. Dasgupta, S., Manna, D. & Basu, G. (2012). FEBS Lett. 586, 1724–1730. Dasgupta, S., Saha, R., Dey, C., Banerjee, R., Roy, S. & Basu, G. (2009). FEBS Lett. 583, 2114–2120. Dubois, D. Y., Blais, S. P., Huot, J. L. & Lapointe, J. (2009). Biochemistry, 48, 6012–6021. Acta Cryst. (2014). F70, 922–927
crystallization communications Eswar, N., Webb, B., Marti-Renom, M. A., Madhusudhan, M. S., Eramian, D., Shen, M.-Y., Pieper, U. & Sali, A. (2006). Curr. Protoc. Bioinformatics, Unit 5.6. doi:10.1002/0471250953.bi0506s15. French, S. & Wilson, K. (1978). Acta Cryst. A34, 517–525. Germain, E., Castro-Roa, D., Zenkin, N. & Gerdes, K. (2013). Mol. Cell, 52, 248–254. Gorrec, F. (2009). J. Appl. Cryst. 42, 1035–1042. Ito, T., Kiyasu, N., Matsunaga, R., Takahashi, S. & Yokoyama, S. (2010). Acta Cryst. D66, 813–820. Kaspy, I., Rotem, E., Weiss, N., Ronin, I., Balaban, N. Q. & Glaser, G. (2013). Nature Commun. 4, 3001. Kawabata, T. (2003). Nucleic Acids Res. 31, 3367–3369. Kern, D., Potier, S., Boulanger, Y. & Lapointe, J. (1979). J. Biol. Chem. 254, 518–524. Lamour, V., Quevillon, S., Diriong, S., N’Guyen, V. C., Lipinski, M. & Mirande, M. (1994). Proc. Natl Acad. Sci. USA, 91, 8670–8674. Lapointe, J., Duplain, L. & Proulx, M. (1986). J. Bacteriol. 165, 88–93. Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674.
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Nureki, O., Vassylyev, D. G., Katayanagi, K., Shimizu, T., Sekine, S., Kigawa, T., Miyazawa, T., Yokoyama, S. & Morikawa, K. (1995). Science, 267, 1958– 1965. Paravisi, S., Fumagalli, G., Riva, M., Morandi, P., Morosi, R., Konarev, P. V., Petoukhov, M. V., Bernier, S., Cheˆnevert, R., Svergun, D. I., Curti, B. & Vanoni, M. A. (2009). FEBS J. 276, 1398–1417. Pflugrath, J. W. (1999). Acta Cryst. D55, 1718–1725. Saha, R., Dasgupta, S., Banerjee, R., Mitra-Bhattacharyya, A., So¨ll, D., Basu, G. & Roy, S. (2012). Biochemistry, 51, 4429–4437. Saha, R., Dasgupta, S., Basu, G. & Roy, S. (2009). Biochem. J. 417, 449–455. Schulze, J. O., Masoumi, A., Nickel, D., Jahn, M., Jahn, D., Schubert, W. D. & Heinz, D. W. (2006). J. Mol. Biol. 361, 888–897. Sekine, S., Nureki, O., Sakamoto, K., Niimi, T., Tateno, M., Go, M., Kohno, T., Brisson, A., Lapointe, J. & Yokoyama, S. (1996). J. Mol. Biol. 256, 685– 700. Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22–25. Vanoni, M. A., Dossena, L., Paravisi, S., Petoukhov, M. & Svergun, D. (2005). HASYLAB Annual Report 2005. http://hasyweb.desy.de/science/annual_ reports/2005_report/part2/contrib/73/14636.pdf. Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242.
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Structural Biology Communications ISSN 2053-230X
Nipa Chongdar,a Saumya Dasgupta,a Ajit Bikram Dattab* and Gautam Basua* a
Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India, and bDepartment of Biochemistry, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India
Correspondence e-mail: [email protected], [email protected]
Received 12 March 2014 Accepted 9 May 2014
Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase from Escherichia coli The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln. To properly understand this distinctive GluRS–tRNA interaction it is important to pursue detailed structure–function studies; however, because of the fact that tRNA– GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure–function correlation studies must also be phylumspecific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNAGlu–GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported.
1. Introduction
# 2014 International Union of Crystallography All rights reserved
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The core function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNAGlu (Kern et al., 1979; Breton et al., 1986), but not all bacterial GluRS restrict their tRNA-glutamylation to only the cognate tRNA (tRNAGlu) (Lapointe et al., 1986; Lamour et al., 1994; Dasgupta & Basu, 2014). In bacteria such as Escherichia coli and Thermus thermophilus that possess glutaminyl-tRNA synthetase (GlnRS), the cognate aminoacylating enzyme for tRNAGln, GluRS exclusively glutamylates tRNAGlu (Kern et al., 1979; Dasgupta et al., 2009; Becker & Kern, 1998). On the other hand, in bacteria that lack GlnRS, such as Bacillus subtilis, Thermosynechococcus elongatus and Mycobacterium tuberculosis, GluRS is equally capable of glutamylating the noncognate tRNAGln (Lapointe et al., 1986; Schulze et al., 2006; Paravisi et al., 2009). In addition, some bacteria, such as Thermotoga maritima, contain two copies of GluRS (GluRS1 and GluRS2) with different specificities for tRNAGlu and tRNAGln (Ito et al., 2010). The diverse tRNAGlx specificities of bacterial GluRS call for structure–function correlation studies that will yield a clear understanding of the mechanisms that control the GluRS–tRNAGlx interaction in bacteria. Because the precise nature of the GluRS– tRNAGlx interaction in bacteria is phylum-specific (Dasgupta et al., 2012; Dasgupta & Basu, 2014), biochemical work performed on the GluRS–tRNAGlx interaction in a bacterium from one phylum may not be compatible with the GluRS structure from a bacterium belonging to a distant phylum. Escherichia coli is a model bacterium, some strains of which are pathogenic. Most biochemical studies have been performed on E. coli GluRS (Eco-GluRS), both in our laboratory (Dasgupta et al., 2009, 2012; Saha et al., 2009, 2012) and in those of others (Sekine et al., 1996; Banerjee et al., 2004; Dubois et al., 2009). Recent reports have demonstrated how Eco-GluRS plays a role in pathogenesis by Acta Cryst. (2014). F70, 922–927
crystallization communications
Figure 1 (a) Homology-modelled structure of Eco-GluRS with colour-annotated domains (1–5). Disordered parts (in 4g6z and annotated as ‘mobile’), the position of Zn and the two surface residues that were mutated in this work (Lys236 and Glu328) are annotated. The Eco-GluRS model was built with superposed multiple templates (PDB entries 2cfo, 4gri and 4g6z) using MODELLER (Eswar et al., 2006). The percentage sequence identities (with Eco-GluRS) are 40.76% (2cfo), 39.49% (4g6z) and 35.45% (4gri), as calculated by ignoring disordered/invisible residues of the templates (residues 103–121 and 379–443 in 4g6z; residues 398–404 and 441–443 in 4gri). (b) Structural alignment of bacterial GluRS using MATRAS (Kawabata, 2003) with DSSP-assigned consensus secondary structure on the top row and residue conservation on the bottom row (colour coded according to the domains in Fig. 1a). Disordered regions (in 4g6z and 4gri) are highlighted in yellow and the corresponding residues are shown in grey. In addition, the proteobacteria-specific Arg266 (Eco-GluRS), the Cys residues in the zinc-binding motif and Lys236/Glu328 are highlighted with red, magenta and blue colours, respectively. The GluRS sequences are denoted by their four-character PDB codes (ECO stands for Eco-GluRS). Two crystal contacts in Bth-GluRS (4g6z), Glu242–Lys243 (c) and Ala334–Ala334 (d), leading to the two surface mutations K236E and E328A in Eco-GluRS.
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crystallization communications inducing multidrug tolerance upon binding to a eukaryote-like serine-threonine kinase (HipA; Germain et al., 2013; Kaspy et al., 2013). Crystal structures of GluRS from all of the species mentioned above, except for B. subtilis and E. coli, are available. The PDB entries are 1j09 (T. thermophilus; Nureki et al., 1995), 2cfo (T. elongatus; Schulze et al., 2006), 2ja2 (M. tuberculosis), 2o5r (T. maritima GluRS1) and 3afh (T. maritima GluRS2; Ito et al., 2010). In addition,
crystal structures of GluRS from Burkholderia thailandensis (PDB entry 4g6z; Baugh et al., 2013) and Borrelia burgdorferi (PDB entry 4gri; T. E. Edwards and M. C. Clifton, unpublished work) are also available. Based on the occurrences of GlnRS in their genomes (Dasgupta & Basu, 2014), the GluRS in B. thailandensis (a GlnRScontaining bacterium) and B. burgdorferi (a GlnRS-lacking bacterium) are expected to be tRNAGln-discriminatory and tRNAGlnnondiscriminatory, respectively. Figs. 1(a) and 1(b) show a homology model for Eco-GluRS (with PDB entries 2cfo, 4g6z and 4gri as templates), its various domains and a structural alignment of all known bacterial GluRS structures and Eco-GluRS. The homology model of Eco-GluRS is not precise enough to reveal the subtleties of GluRS–tRNAGlu interaction in E. coli owing to the absence of some specific features of Eco-GluRS in the template structures. For example, the proteobacterial-specific residue Arg266, which is present in a tRNA-interacting loop of Eco-GluRS (Dasgupta et al., 2012), is absent in all known bacterial GluRS structures except for that of B. thailandensis GluRS (Bth-GluRS; see Fig. 1b). The structure of Bth-GluRS (Fig. 1b), however, is unsuitable for detailed structural studies on Eco-GluRS not only because the SWIM-like domain of Bth-GluRS (100C-x-M-xn-W-x-P129) lacks the characteristic ‘C-x-C-xn-C-x-H’ zinc-binding motif of Eco-GluRS (Banerjee et al., 2004), but also because a long stretch of residues (103–121) encompassing the zinc-binding motif are disordered in the crystal structure 4g6z. Similarly, residues 379–443 in the functionally important Cterminal domain (domain 5) are also disordered in 4g6z. In order to overcome these shortcomings, it is imperative that the structure of Eco-GluRS be determined. Here, we report the expression, purification and preliminary crystallization screening of an engineered version of Eco-GluRS in which two surface residues, Lys236 and Glu328, were mutated to facilitate crystal packing.
2. Methods 2.1. Design of surface-engineered Eco-GluRS
Figure 2 (a) SDS-PAGE [8%(w/v)] analysis of dd-GluRS: lane 1, (6His)-(SUMO2 tag)(Gly)-(dd-GluRS) (64.7 kDa); lane 2, (Gly)-(dd-GluRS) (53.8 kDa) and 6HisSUMO2 (10.9 kDa) after SENP2 digestion; lane 3, purified fraction of (Gly)-(ddGluRS); lane 4, molecular-weight markers (labelled in kDa). (b) CD spectra of 5 mM wild-type Eco-GluRS and dd-GluRS in 50 mM pH 7.5 phosphate buffer containing 100 mM KCl at 298 K. (c) The time course of product (Glu–tRNAGlu) formation during glutamylation of Eco-tRNAGlu by wild-type Eco-GluRS and ddGluRS.
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Eco-GluRS is known to resist crystallization (Vanoni et al., 2005). Previous attempts to crystallize Eco-GluRS in our laboratory were also unsuccessful. A number of factors might have contributed to this failure, including inter-domain dynamics, but this can be ruled out for Eco-GluRS since quite a few homologous GluRSs from other bacteria have successfully been crystallized. Another factor responsible for the observed resistance of Eco-GluRS crystallization could be unfavourable crystal contacts. In order to identify a subset of EcoGluRS residues that might be hindering lattice formation, we compiled a list of residue pairs involved in pairwise crystal contacts (pccs) in an evolutionarily close homologue (Bth-GluRS; PDB entry 4g6z). Using a multiple sequence alignment of bacterial GluRS, we pruned the pccs by removing (i) residues that were identical between Eco-GluRS and Bth-GluRS and (ii) residues that were mostly conserved in the multiple sequence alignment. Physical interactions between all the remaining residue pairs in the pruned pccs were visually inspected in the Bth-GluRS structure and compared with the Eco-GluRS model (built with 4g6z as the template). This allowed us to identify two energetically favourable pairs in 4g6z that were unfavourable in Eco-GluRS. The first pair involves residues Glu242 and Lys243 in 4g6z, which form an electrostatically favourable salt bridge (Fig. 1c), but the interaction between corresponding residues in Eco-GluRS (Lys236–Lys237) is electrostatically unfavourable (Fig. 1c). The second residue pair Ala334–Ala334 in 4g6z (Fig. 1d) is electrostatically neutral whereas the corresponding pair in EcoGluRS (Glu328–Glu328) (Fig. 1b) is electrostatically unfavourable. Acta Cryst. (2014). F70, 922–927
crystallization communications In order to remove these two plausible unfavourable crystal contacts, we introduced two mutations (K236E/E328A) in Eco-GluRS and attempted crystallizing the double mutant (dd-GluRS).
mutations were confirmed by DNA sequencing (Applied Biosystems).
2.2. Cloning of Eco-GluRS in pETSUMO2
2.3. Overexpression and purification
The GluRS-encoding gene of E. coli was amplified from plasmid DNA PLQ7619 using appropriate primers. The amplified product was cloned in a pETSUMO2 vector (Datta & Wolberger, unpublished work) using an In-Fusion PCR Cloning Kit (Clontech). The resulting plasmid coded for Eco-GluRS with an N-terminal 6His-SUMO2 tag and a Gly residue [(6His)-(SUMO2 tag)-(Gly)-(Eco-GluRS)]. K236E and E328A mutations were introduced into Eco-GluRS by PCR-based site-directed mutagenesis using appropriate primers. The
Cultures of E. coli Rosetta2 (DE3) strain transformed with the recombinant plasmid were grown in Circlegrow (MP Biomedicals) medium supplemented with 100 mg ml1 ampicillin at 310 K until the OD600 reached 1; protein expression was induced with 0.5 mM isopropyl -d-1-thiogalactopyranoside (IPTG) for 16 h at 288 K. The cells were harvested by centrifugation (5000g for 10 min) followed by resuspension in ice-cold lysis buffer (20 mM sodium phosphate pH 8, 500 mM NaCl, 5 mM MgCl2, 10 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride and DNase I) and lysed in a microfluidizer (Microfluidics) at 277 K. The crude lysate was clarified by centrifugation (20 000g for 45 min at 277 K) followed by filtration through a 0.22 mm membrane (Millipore). The recombinant GluRS was purified from the clarified lysate using a 5 ml HisTrap HP affinity column (GE Healthcare) in 20 mM phosphate buffer pH 8 containing 400 mM NaCl, 20 mM ZnCl2 under a linear gradient of imidazole (20– 400 mM). The purified fusion protein [(6His)-(SUMO2 tag)-(Gly)(Eco-GluRS)] was dialyzed in 20 mM sodium phosphate buffer pH 8 containing 150 mM NaCl, 20 mM ZnCl2, 10 mM -mercaptoethanol for 16 h at 277 K, during which the 6His-SUMO2 tag was cleaved by adding SENP2 protease (3 mg SENP2 was added to 150 mg fusion protein; lanes 1 and 2 of Fig. 2a). The mixture was passed through another HisTrap HP affinity column (GE Healthcare) to remove the (6His)-(SUMO2) tag and SENP2. The flowthrough, containing the untagged GluRS, was estimated to be of >95% purity as judged by SDS–PAGE followed by Coomassie staining (lane 3 of Fig. 2a). It should be noted that in the absence of the additional glycine between the 6His-SUMO2 tag and the Met1 of Eco-GluRS, cleavage efficiency was substantially reduced (40–50%) under identical conditions, even with a higher SENP2:protein ratio [increased to 0.1:1(w:w) from 0.02:1(w:w)]. All the chromatographic steps were performed ¨ KTA Avant liquid-chromatography system (GE Healthusing the A care). Purified (Gly)-(Eco-GluRS) was dialyzed in storage buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM -mercaptoethanol, 50 mM ZnCl2), concentrated to 35 mg ml1 using centrifugal concentrators of molecular-weight cutoff 30 kDa (Vivaspin 20, Sartorius AG) and stored at 193 K after flash-freezing in liquid nitrogen.
2.4. Secondary-structure analysis and glutamylation assay
Figure 3 (a) A crystal of dd-GluRS obtained using the hanging-drop vapour-diffusion method (see x2). (b) A typical diffraction pattern of the GluRS crystal (oscillation ˚ from the centre). image) annotated with resolution arcs (8, 5, 4 and 3 A
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Changes in the secondary-structure contents of the wild-type and mutant Eco-GluRS were assessed from circular-dichroism (CD) spectra (200–260 nm) of 5 mM protein (Fig. 2b) recorded in a J-815 CD spectropolarimeter (Jasco) with a 2 mm cuvette in 50 mM phosphate buffer pH 7.5 containing 100 mM KCl at 298 K. The enzymatic activities of wild-type Eco-GluRS and dd-GluRS were determined by in vitro aminoacylation assays where the rate of product ([3H]-l-Glu–tRNAGlu) formation was monitored as a function of time. The reactions were carried out with 5 mM E. coli tRNAGlu in 50 mM HEPES pH 7.5, 0.1 mM unlabelled l-Glu, 16 mM MgCl2, 2 mM ATP, 0.8 mM -mercaptoethanol and [3H]-l-Glu (0.5 ml of stock per 100 ml of assay mixture) at 310 K as described previously (Dasgupta et al., 2009). Protein concentrations used per assay point were 5.4 nM. Chongdar et al.
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crystallization communications Table 1 Data collection and processing statistics. Values in parentheses are for the outermost shell. Diffraction source ˚) Wavelength (A Temperature (K) Exposure time (s) Space group ˚ , ) Unit-cell parameters (A
RU 300 [Blue optics] 1.54178 110 300 C2 a = 213.05, b = 61.52, c = 102.74, = 96.25 29.89–3.30 (3.42–3.30) 67555 20231 99.8 (100) 3.34 (3.38) 8.8 (1.5) 2.19 6.2 (49.0) 3.17 2
˚) Resolution range (A Observed reflections Unique reflections Completeness (%) Multiplicity hI/(I)i Average mosaicity ( ) Rmerge† (%) ˚ 3 Da1) VM (A Molecules per asymmetric unit † Rmerge =
P
hkl
P
i
jIi ðhklÞ hIðhklÞij=
P
hkl
P
i Ii ðhklÞ.
2.5. Crystallization and X-ray diffraction data collection
Initial crystallization screenings were performed using commercially available screens (Index and PEGRx; Hampton Research) and Morpheus (Gorrec, 2009) at 293 K in 24-well XRL plates (Molecular Dimensions) using the hanging-drop vapour-diffusion method. Morpheus condition 90 [0.1 M MOPS/HEPES–Na pH 7.5, 0.02 M each of l-Glu.Na, dl-Ala, dl-Lys.HCl, Gly and dl-Ser, 10%(w/v) PEG 8000, 20%(v/v) ethylene glycol] yielded crystal clusters. The crystallization condition was further optimized by varying the pH (7.3–7.9), the concentrations of PEG 8000 [10–14%(w/v)] and ethylene glycol [14–28%(v/v)] and the protein:crystallization cocktail ratio [1:1–1:2(v:v)]. Single crystals suitable for diffraction (Fig. 3a) were obtained from drops prepared with 1 ml 20 mg ml1 dd-GluRS and 1.2 ml of the optimized crystallization cocktail [0.1 M MOPS/ HEPES–Na pH 7.7, 0.02 M each of l-Glu.Na, dl-Ala, dl-Lys.HCl, Gly and dl-Ser, 14%(w/v) PEG 8000, 22%(v/v) ethylene glycol]. Crystals grew to a size of about 300 100 50 mm in two weeks and ˚ (Fig. 3b). typically diffracted to a resolution of 3.5 A Prior to X-ray diffraction data collection, the crystals were soaked in mother liquor containing an increasing concentration of ethylene glycol, from 22% to 32%, in 2% steps with about 1 min of soaking at each step. A few initial images were collected and processed with d*TREK (Pflugrath, 1999) to identify the space group and unit-cell parameters. Final X-ray diffraction data were collected in-house at 110 K in steps of 1 over 170 total rotation using a Rigaku RU300 generator with a Cu anode and an R-AXIS IV++ image-plate detector. The data were integrated, scaled and merged using d*TREK. The crystals belonged to the monoclinic space group C2, ˚, = with unit-cell parameters a = 213.05, b = 61.52, c = 102.74 A 96.25 . The data statistics are summarized in Table 1. The merged intensity data were imported into the CCP4 suite (Winn et al., 2011) and converted to structure-factor amplitudes using CTRUNCATE (French & Wilson, 1978). The solvent content was estimated from the Matthews coefficient (Matthews, 1968) using a molecular mass of 53.8 kDa. Molecular-replacement phasing was attempted with either Phaser (McCoy et al., 2007) or MOLREP (Vagin & Teplyakov, 2010).
3. Results and discussion Based on the crystal packing of Bth-GluRS structure (PDB entry 4g6z), the designed double mutant (K236E/E328A) of Eco-GluRS (dd-GluRS) was successfully crystallized. The corresponding wild-
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type GluRS failed to crystallize under similar conditions. The secondary structure contents of dd-GluRS and the wild-type protein were indistinguishable, as judged from CD studies (Fig. 2b). The enzymatic activities (rate of formation of the product Glu–tRNAGlu) of wild-type Eco-GluRS and dd-GluRS were also very similar as indicated by the slopes of product formation at early time points (Fig. 2c). Therefore, not only did the engineered GluRS crystallize but the mutations that enabled crystallization of the protein did not affect its overall structure or the function. The volume of the asymmetric unit in the dd-GluRS crystal was compatible with the presence of either two or three monomers in the asymmetric unit, with a volume per unit molecular weight of the ˚ 3 Da1 and a calculated solvent content protein (VM) of 3.02–2.03 A of 59.65–39.48% (Matthews, 1968). Molecular-replacement trials performed using either PDB entry 2cfo (GluRS from T. elongatus; Schulze et al., 2006) or 4g6z (B. thailandensis GluRS; Baugh et al., 2013) as search models consistently showed two monomers in the asymmetric unit. However, neither of the search models yielded electron-density maps that were good enough to permit further building and refinement of the model with reasonable R factors. The two domains of GluRS (see Fig. 1) show rigid-body dynamics in superimposed structures of homologous GluRS. To address this issue, we also tried molecular replacement by defining two domains from PDB entry 2cfo (2–384 and 385–485) as separate ensembles in a single search. Although this improved the map quality, it was still not possible to complete the structure. The poor molecular-replacement phasing could be due to low resolution and/or poor data quality. To circumvent this problem, we are currently pursuing further optimizations to obtain higher resolution data with options of including experimental phasing techniques. Efforts to crystallize Eco-GluRS bound to ligands (tRNAGlu and ATP) are also under way. In summary, we have successfully crystallized Eco-GluRS by introducing two point mutations. dd-GluRS crystallized in space group C2, different from the space group (P41212) of the template (4g6z) used for optimizing the crystal contacts in Eco-GluRS. Nonetheless, the fact that the engineered crystal contacts played a role in successful crystallization can certainly be concluded from the fact that the wildtype protein did not crystallize under identical conditions. The mutated residues therefore either play a role in crystal nucleation or stabilize the final crystal lattice. The latter can only be confirmed once the structure has been solved, although all preliminary molecularreplacement solutions point towards crystal contacts by K236E and E328A. GB acknowledges financial supported by a Wellcome Fellowship. We thank Adaitya SENP2 protease and Maura carefully.
support from CSIR, India. ABD is Trust–DBT Alliance Intermediate Behera for his help in preparing the Hurley for reading the manuscript
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