Leucyl-tRNA synthetase from Thermus thermophilus . Purification and some properties of the crystallizing enzyme

Leucyl-tRNA synthetase from Thermus thermophilus (LeuRSTT) was purified to homogeneity using a five-step purification procedure. The enzyme was characterized and crystallized. Molecular mass determinations of the native and denatured proteins indicate monomeric structure of LeuRSTT with the molecular mass of about 101 kDa, The protein obtained is remarkably thermostable and retains 97 % of its initial aminoacylation activity after 1 hour of incubation at 88 °С. Crystals of LeuRSTT were obtained from ammonium sulfate solution by the vapour diffusion techniques. The crystals quality was improved by crystallization from the precipitate.

Introduction.Aminoacyl-tRNA synthetases (aaRSs) catalyze the specific esterification of an amino acid to the З'-end of its cognate tRNA.Through a two-step reaction, aaRSs use first ATP to form the activated intermediate, aminoacyl-adenylate, and then transfer the amino acid to the З'-end of their cognate tRNA.The reaction is highly specific, although in some cases, to ensure the fidelity of the genetic code translation, the aaRS relies on an editing activity to hydrolyze misactivated amino acids which are similar to the cognate amino acid [1,2].The family of twenty aaRSs are divided into two distinct classes of ten enzymes each on the basis of the primary and tertiary structures information [3,4 ].Also, all tRNA molecules can be classified into two types according to the length of the variable arm.Type I tRNAs (most of tRNA molecules) have a short variable arm com prising four or five nucleotides.Type II tRNAs (tRNA 1 ^ from eubacteria and organelles of lower eukaryotes, tRNA Uu and tRNA Ser ) have a long va riable arm composed of more than ten nucleotides.
Leucyl-tRNA synthetase (LeuRS) is a monomeric class I enzyme which recognizes type II tRNA.It was shown by mutational studies that Escherichia coli LeuRS recognized tRNA Uu in a manner different from the standard recognition mode [5].Only the discriminator base A73 is required for the basespecific recognition by LeuRS while the anticodon, the variable stem loop and the acceptor stem sequences are not essential for the specific recognition by LeuRS.To define the recognition mode and structural identity elements of tRNA 1 *" by LeuRS as well as to get detailed information on substrate specificity and enzyme mechanism it is necessary to determine the crystal structure of the enzyme alone and in the complex with substrates.So far crystal structures of sixteen from twenty different synthetases have now been determined together with a variety of substrate complexes [6,8 ].LeuRS is one of four aaRSs which crystal structure is not known yet.Here, we report the purification, some properties and crystallization of LeuRSTT.
Purification of T. thermophilus LeuRS and tRNA Uu .The T. thermophilus (strain HB8 or HB27) cells were harvested by centrifugation, washed and disrupted by a French Press in 100 mM Tris-HCl buffer, containing 2 mM dithiothreitol, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, com pleteTM (1 tabl./25ml extraction buffer), 5 mM MgCl 2 and 1 mM NaN 3 .The extract was centrifuged for 2 hours at 105000 g and the supernatant was subjected to 30-65 % ammonium sulfate fractio nation.The precipitate containing LeuRS activity was dissolved in 20 mM Tris-HCl buffer, pH 7.5 con taining 5 mM MgCl 2 , 0.1 mM phenylmethylsulfonyl fluoride, 2 mM DTT, 1 mM NaN 3 (buffer A), dialyzed against buffer A and absorbed on a DEAE-Sepharose column (5 x 55 cm) equilibrated with buff er A. The LeuRS was eluted with 2 x 2.5 L linear gradient of 0.03 M to 0.3 M sodium chloride in buffer A. The fractions containing LeuRS activity were salted out by ammonium sulfate (50 % saturation) and applied to a Toyopearl HW-65 column (3 x x 80 cm) equilibrated with 40 % of ammonium sul fate in buffer A. The proteins were eluted with 2x2 L gradient of 40-10 % saturation ammonium sulfate in buffer A. Fractions containing LeuRS activity were pooled, dialyzed and chromatographed on a hydroxylapatite column (3 x 40 cm) with 2 x 2 L potas sium phosphate buffer linear gradient from 0.01 M to 0.25 M (pH 7.9).After dialysis in buffer A of the active fractions the enzyme was loaded on a Heparin Sepharose CL-6B column (1 x 40 cm).A 1.0 L linear 0-0.25 M KC1 gradient in buffer A was used to elute the LeuRS.All steps of the enzyme purification were carried out at 4 °С T. thermophilus tRNA Uu was purified from bulk tRNA using BD-cellulose, anion-exchange DEAE 5PW (HPLC, «Весктап», USA) and C8 reversephase chromatography (I.Krikliviy, D. Rozhko and M. Tukalo, unpublished results).
The molecular mass determination.
The mo lecular mass of the enzyme was determined by gel filtration on a column (1.1x72 cm) of Sephadex G-200 and by PAGE under native and denaturing conditions.Under native conditions it was conducted in 5 % and 6 % polyacrylamide gels.Under de naturing conditions it was conducted in 12.5 % polyacrylamide gel in the presence of 0.1 % SDS as described by Laemmli [9].
Measurements of thermal stability of LeuRS.The heat stability of the aminoacylation activity of LeuRSTT was analyzed by incubating 100 /Л of the enzyme solution (1.5 mg/ml) in 100 mM Na-Hepes, pH 7.3 containing 15 mM MgCl 2 with or without the small substrates at the indicated concentration.At various times, 10-/Л aliquots were removed, diluted in cold enzyme dilution buffer containing 100 mM Na-Hepes, pH 7.3, 2 mM DTT, 10 % glycerol, 15 mM MgCl 2 and 5 mg/ml BSA.The remaining aminoacylation activity was determined by initial rate measurements as described above.
Crystallization procedure.Crystallization trials were conducted at different constant temperatures (6,12, and 20 °С) using the hanging-drop vapor-dif fusion method.The drops of 4-10 jul were allowed to equilibrate against 0.8 ml reservoir solution.Con ditions such as the nature of the precipitant, the composition and pH of the buffer and the con centration of the protein were varied.
Results and Discussion.LeuRSTT was purified from both HB8 and HB27 strains to homogeneity using a five-step purification procedure (Table 1).The final yield was approximately 20 mg of the pure enzyme from 520 g cells with a specific activity of 2212 U/mg (1 U of the enzyme catalyzes the formation of 1 nmol leucyl-tRNA/min at 65 °С).Gel filtration gives M r of 100 kDa.PAGE under native conditions reveals an apparent M r of 102 kDa.SDS/PAGE shows a single band corresponding to a polypeptide chain of about 101 kDa.The kinetic constants of the aminoacylation reaction catalyzed by

Table I Purification of leucyl-tRNA synthetase from T. thermophilus HB8
•Obtained from 520 g cells.

Table 2 Kinetic constants of aminoacylation reaction catalyzed by LeuRS from T. thermophilus at 65 °С
LeuRSTT for ATP, leucine and tRNA Uu are reported in Table 2.There is no significant difference in K m and к ш values of LeuRSTT for all three substrates compared with those of LeuRS from E. coli [10].The optimal temperature for the tRNA aminoacylation reaction catalyzed by LeuRSTT is about 70 °С for tRNA Uu from T. thermophilus and about 65 °С for tRNA Uu from E. coli (Fig. 1).The thermal stability of the enzyme was investigated in the range from 50 to 90 °С.The enzyme is remarkably thermostable.It retains 100 % of its ability to aminoacylate tRNA after 1 h of incubation at 85 °С (data not shown) and about 97 % at 88 °С.ATP and leucine alone do not affect the thermal inactivation of the enzyme at 88 °С.Surprisingly, when ATP and leucine or ATP, leucine and inorganic pyrophosphate are present together significant inactivation of LeuRS occurs after 1 h at 88 °С (Fig. 2).In contrast, the protection of GlyRS from thermoinactivation by ATP, glycine or glycyladenylate was shown for T. thermophilus GlyRS [11], ATP or tyrosyladenylate for TyrRS, histidine or histidyladenylate for HisRS and prolyladenylate for ProRS from T. thermophilus (Yaremchuk et al., unpublished results).In all these cases the protection of the aaRS from thermal inactivation by substrates correlates with conformational changes of the enzyme promoted by substrates binding ( [11], Cusack S., Yaremchuk A. and Tukalo M., unpublished results).
To study the mechanism of amino acid re cognition and activation and the specific recognition of cognate tRNA by T. thermophilus LeuRS we tried to crystallize this enzyme alone and in complex with the substrates.Initial screening of crystallization con ditions was conducted using a sparse matrix sampling reagents from Hampton Research (Crystal screen 1 and 2, Grid screens including ammonium sulfate, polyethylene glycol 6000, sodium chloride, 2-methyl-2,4-pentanediol and Crystal screen cryo formulation) without any success.Precipitants alone, such as ammonium sulfate, PEG, sodium formate, sodium citrate, sodium chloride and MPD at different con centrations over a broad range of pH and protein concentration did not yield crystals suitable for X-ray structure determination.Extremely thin needle-like crystals were obtained from 48 % ammonium sulfate solution at pH 7.5-8.3 at 6 °С and were not of suitable dimensions for diffraction experiments.Not well-shaped crystals (Fig. 3, a) and diffracting to 9 A resolution were obtained at 20 °С from 38-40 % ammonium sulfate solution at pH 7.5 in the presence of 5 % glycerol after two-three weeks and are very poorly reproducible.SDS/PAGE shows degradation of the enzyme after one week at 20 °С and even crystals contain two polypeptide chains with molecular mass of about 100 and 96 kDa (data not shown).Betterquality crystals of LeuRSTT were obtained under the following conditions: to, prevent protein degradation 10/Л drops containing 8-10 mg/ml LeuRS in 40 mM Tris-HCl, pH 7.5, 2 mM DTT, 10 mM MgCl 2 , 1 mM NaN 3 , and 15 % ammonium sulfate were equilibrated for 12-14 hours at 6 °С against 800 ml reservoir solution containing 54-58 % ammonium sulfate in 100 mM Tris-HCl, pH 7.5.After rapid precipitation of the macromolecules the drops were transferred to 12 °С and ammonium sulfate concentration was decreased to 40-42 %.Under these conditions well-shaped crystals appeared in two days and grew from precipitate rapidly over a 4-5 days period to a maximum of 0,8 mm in the longest dimension (Fig. 3, b).After stabilization for one week against 48 % saturated ammonium sulfate in 100 mM Tris-HCl, pH 7.5 crystals were mounted in quartz capillaries with a small amount of reservoir solution for X-ray dif fraction measurements.These crystals diffract to 3.5 A resolution.The protein from washed and solubilized crystals as well as the protein used for crystallization was verified as full-length LeuRSTT by SDS/PAGE and enzymatic ability to aminoacylate tRNA.X-ray diffraction analysis of these crystals is under way.Small crystals of LeuRSTT in complex with tRNA Uu were obtained using ammonium sulfate as a precipitant but they were not of suitable dimen sions for X-ray analysis.

Fig, 3 .
Fig, 3. Crystals of LeuRSTT: a -not well-shaped crystals and diffracting to 9 A resolution; b -crystalls obtained by crystallization from precipitate and diffracting to 3.5 A resolution