THE J O U R N A I . zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAOF BIOLOCICAI. CHEMISTRY V d zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA258, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBANo. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA13, Issue of Ju ly IO, pp. 8429-8435.1983 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAi ’nnfed in U.S.A.

Crystallization of a tRNA. Aminoacyl-tRNA Synthetase Complex CHARACTERIZATION AND FIRST CRYSTALLOGRAPHIC DATA*

(Received for publication, February 14, 1983)

Bernard Lorber, Richard Giege, Jean-Pierre Ebel, Carmen BerthetS, Jean-Claude Thierry, and Din0 Moras zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFrom the Laboratoires de Biochimie et Cristallographie Eiologique, Institut de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, I5 Rue R. Descartes, F-67084 Strasbourg Cedex, France

A complex formed between the dimeric aspartyl- tRNA synthetase from yeast (Mr = 125,000) and two molecules of its cognate yeast tRNAAsP (Mr = 24,160) was crystallized using ammonium sulfate as the precip- itant. The crucial parameter which governs a success- ful crystallization is the enzyme. tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, bi- ological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent con- tent is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crgstals is suitable for x-ray diffraction studies up to 7-A resolution.

The first key step in protein synthesis is the correct attach- ment of amino acids to tRNAs by their cognate aminoacyl- tRNA synthetases (1). This highly specific reaction implies a sophisticated nucleic acid-protein recognition mechanism since for each amino acid not only one enzyme but several isoacceptor tRNAs exist and still each synthetase accurately aminoacylates its macromolecular substrates in the presence of ATP and the corresponding amino acid.

A basic understanding, at the molecular level, of the reac- tion pathway requires the knowledge of the three-dimensional structure of the different components in their free and com- plexed states. Crystallography is a powerful tool for that purpose. Up to now the structures of five tRNA molecules have been solved at various resolutions (2-9). The tertiary structures of one synthetase and of an active t r p t i c fragment are also known at resolutions better than 3 A from x-ray studies (10,11). So far no structural investigation of a complex between a synthetase and its cognate tRNA by diffraction methods has been possible since no crystals were available

* This work was supported by grants from the Centre National de la Recherche Scientifique (Action Thematique Programmee: Struc- ture Tridimensionnelle des Proteines), the Delegation Generale i la Recherche Scientifique et Technique, and the Ministere de la Re- cherche et de 1’Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to Professor E. Lederer on the occasion of his 75th birthday.

$ Permanent address, European Molecular, Biology Laboratory, c/o Institut Laue Langevin, 156X. F-38042 Grenoble Cedex, France.

~~ -~

despite numerous attempts. After the first successful crystallization of yeast tRNAAsP

(12), the focus was on the aspartic acid system from yeast and suitable crystals of the cognate aspartyl-tRNA synthetase were soon obtained using ammonium sulfate as the precipitant (13). The fact that both molecules, the enzyme and its tRNA substrate, were crystallized under similar high salt conditions raised the possibility of using the same precipitant to CO-

crystallize the molecules in their complexed state. Although high ionic strength was thought to hamper complex formation, it was shown that the aminoacylation reaction could occur at high ammonium sulfate concentrations (14). After this posi- tive result crystallization attempts were successfully carried on. The results of these experiments including the character- ization of the macromolecular components within the crystals and the first crystallographic data are reported in this paper. Preliminary and partial results of this work have been pub- lished as a short communication (15).

MATERIALS AND METHODS

General

Ammonium sulfate was purchased from Merck (Darmstadt, Ger- many). A standard saturated solution (100% of saturation) was pre- pared at 20 “C and used for dilutions throughout all experiments reported here. HEPES,’ MES, cacodylate (acid and sodium salt), Tris (base and hydrochloride), and dithioerythritol were from Sigma. Spermine was from Fluka (Buchs, Switzerland). Acrylamide and methylene bisacrylamide especially purified for electrophoresis were obtained from BDH Chemicals Ltd. (Poole, England). Molecular weight standard protein mixtures were from Pharmacia (France S. A,) and Sigma. ~-[‘~C]Aspartic acid was purchased from the Com- missariat a 1’Energie Atomique, Saclay, France. Before use, Visking dialysis tubing was boiled in 5% (w/v) sodium bicarbonate and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA5 mM EDTA, rinsed in distilled water, and stored in 50% ethanol. All solutions used for crystallization were prepared with quartz-distilled water and filtered on Millex GS (0.22 pm) filter units from Millipore Corp. (Bedford, MA).

Yeast Aspartyl-tRNA Synthetase and tRNAAsp

Aspartyl-tRNA synthetase, a dimer of M , 125,000, was purified from Saccharomyces cereuisiae (bakers’ yeast) as previously described (13, 16, 17). The enzyme had a specific activity of 600 units/mg (1 unit yields 1 nmol of aspartyl-tRNAASP/min at 37 “C under the standard conditions described below). The apparent low value of the specific activity of the enzyme is the consequence of a nonsaturating amount of aspartic acid in the assay mixture due to the weak affinity of the amino acid for the synthetase. tRNAASp (M, = 24,160) was purified from crude tRNA from brewers’ yeast (Boehringer Mann- heim) as previously reported (18, 19). The tRNA accepted 1,590 pmol of aspartic acidiabsorbance unit a t 260 nm. The choice of two

The abbreviations used are: HEPES, N-2-hydroxyethylpipera- zine-Nf-2-ethanesulfonic acid; MES, 2-(N-morpholino)ethanesul- fonic acid.

8429

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8430 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBACrystals zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof tRNA. Aminoacyl-tRNA Synthetase Complex zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAdifferent yeast strains for purifying tRNA and enzyme was dictated by practical considerations. Large scale purification of the enzyme was possible using the commercial bakers' yeast from the Societ6 Fabrique Alsacieucie de Levuse et d'Alcoal, Strasbourg; crude tRNA from brewers' yeast was used since this tRNA possesses intact CCA zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA3 ' termini in contrast to the crude bakers' yeast tRNA (17). After purification the enzyme was stored a t -20 "C as a concentrated stock solution of 20 mg. ml" in 50 mM potassium phosphate buffer a t pH 7.2 containing 5 mM 2-mercaptoethanol, 0.1 mM EDTA, and 50% glycerol; the tRNA was stored as dry pellets (12).

metric measurements on ZEISS M4 QIII or ZEISS DMR 10 spectro- Macromolecule concentrations were obtained from spectrophoto-

photometers. The extinction coefficient of aspartyl-tRNA synthetase was determined by amino acid composition (20, 21), colorimetric titration according to Lowry et al. (22), and dry weight measurements; a mean value of E$~%I = 0.60 * 0.06 was obtained. Thus, one absorbance unit at 280 nm corresponds to (1.67 k 0.17) mg.ml". In the case of tRNAAsp the extinction coefficient was determined by phosphorus titration (23); one optical density unit a t 260 nm was taken to correspond to 40 pg.ml" in 1-cm path length cells.

Crystallization Procedure

Crystallization was conducted by the vapor diffusion method using the sitting drop technique (24). Glass depression plates were silicon- ized with Sigmacote SL-2 (Sigma) and mounted in transparent plastic boxes. The methods were close to those described for the tRNA (12) and the enzyme (13) alone.

Before crystallization the enzyme was dialyzed at 4 "C against buffers as indicated, containing ammonium sulfate a t 40% of satu- ration. Significant amounts of the enzyme can be irreversibly ad- sorbed on the dialysis tubing and are then lost. This effect is overcome when the dialysis is conducted in the presence of 0.1% (w/v) 1-0- octyl-/.-D-glucopyranoside (Serva, Heidelberg, Germany) in tubing previously treated with the detergent. It was verified that this treat- ment does not affect the activity of the enzyme. Dialyzed enzyme solutions were centrifuged 30 min a t 9000 X g. Before use, tRNA pellets (5 mg) were dissolved in 200 p1 of quartz-distilled water. Crystallization drops (30 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBApl) were prepared by mixing 15 pl of complex solution made of 12 pl of aspartyl-tRNA synthetase in its dialysis buffer and 3 p1 of tRNAAsP in water, with 15 pl of dialysis buffer containing, if indicated, various additives.

Tris-HC1 buffer a t pH 7.8 containing 0.18 mM EDTA, 0.18 mM The hest crystals were obtained in a starting solution of 45 mM

dithioerythritol, ammonium sulfate a t 36% of saturation with tRNAAsp(l,2 to 2.0 mg.ml"), and aspartyl-tRNA synthetase (3.0 to 5.0 mg.ml") in a stoichiometric ratio of two tRNA molecules to one enzyme molecule. Crystallization drops were equilibrated a t 4 "C against 25-ml reservoirs of ammonium sulfate at 52% of saturation. Numerous crystallization conditions were tested as indicated under "Results" by varying the nature, concentration, and pH of the buffer, the concentration of enzyme, and the stoichiometry of the complex, polyamines, divalent cations, reducing agents, etc.

Crystal Handling

When crystal growth was achieved, the crystals were stabilized by increasing slowly the concentration of the precipitant in the reservoir by addition of a saturated ammonium sulfate solution to reach a final concentration of 70%. At this stage, crystals can be used for biochem- ical and x-ray investigations. For biochemical measurements, crystals were washed several times with fresh mother liquor containing am- monium sulfate at 70% of saturation in order to eliminate noncrys- tallized protein and nucleic acid. Just before use mother liquor was removed and crystals were dissolved in a small volume of buffer solution containing 50 mM MES-KOH at pH 6.8, 120 mM KC1, and 10 mM glutathione. Aliquots were taken for the analyses. Crystal size was measured under the microscope on crystals outside their mother liquor so that optical effects due to the crystallization droplet were eliminated. For x-ray investigations crystals were mounted in Lin- demann glass capillaries (Hilgenberg Glass, Malsfeld, Germany) with a droplet of mother liquor and sealed with wax.

Biological Actiuity Assay

Aspartyl-tRNA synthetase was assayed by the aminoacylation reaction of tRNAABp from yeast. Standard assay conditions were optimized for the detection of very low quantities of enzyme. The reaction mixture contained 50 mM MES-KOH buffer at pH 6.8, 30 mM KCI, 15 mM MgClz, 10 mM ATP, 2.5 mM glutathione, 120 mM

(NHJ2S04, 0.1 mM ~-[ '~C]aspart ic acid (200 mCi.mmol"), and an excess of tRNAAsp (=5 p ~ ) . Assays were initiated by the addition of the appropriate amount of enzyme to the reaction mixture. For the test of synthetase activity, crystals were dissolved in a buffer solution, as indicated, deprived of ATP, MgCl?, aspartic acid, and tRNA, and assays were initiated with aliquots of this material. Incubations were a t 37 "C for short times (30 s to 5 min) in order to ensure the linearity of the aminoacyl-tRNA formation when specific activities of the enzyme are measured or to eliminate parasite reactions which occur at a much lower rate when plateau values of tRNA charges are measured in the presence of high amounts of enzyme (25). Aliquots of 40 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAp1 were withdrawn from the incubation mixtures and deposited on Whatman No. 3MM paper discs. The radioactive aspartyl-tRNA was precipitated on the discs in 5% trichloroacetic acid and washed twice by the precipitant; aft,er two additional washes with ethanol, the discs were dried and counted by liquid scintillation.

We note that under these assay conditions (short incubation time and absence of bovine serum albumin in the reaction mixture), the nonspecific fixation of aspartic acid on aspartyl-tRNA synthetase was negligible (25).

Electrophoretic Analyses

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for Proteins-For routine analysis of single crystals, polyacrylamide gels (11% (w/v) T, 2.7% (w/w) C, 1% (w/v) sodium dodecyl sulfate) were prepared according to established procedures (26, 27). The thin ver- tical slab gels (160 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAX 140 X 0.5 mm) were run at 20 "C for 14 h a t a constant 150 V. Bromphenol blue was used as front marker and was allowed to reach 1.5 cm over the bottom of the gel a t the end of the migration. All protein samples, brought up to the same volume (15 pl), were mixed with the same volume of twice concentrated disso- ciating buffer solution (26) and heated for 2 min a t 100 "C in a water bath. The gels were stained by classical techniques using Coomassie brilliant blue (G-250) (Serva), PAGE blue 83 (BDH), or silver stain (28).

Gel Electrophoresis for tRNA-Experiments were conducted in vertical slabs of the same dimension as those used for proteins. Polyacrylamide gels (12% (w/w) T, 5.3% (w/w) C) were polymerized in the presence of 8 M urea and run with a Tris borate buffer at pH 8.3 (Tris base 10.8 g . liter-', boric acid 5.5 g . liter", and EDTA 0.93 g.liter-I). Migration was for 5 h at constant voltage (200 V) at 20 "C, with bromphenol blue as marker dye. Gels were stained routinely with Stains All from Eastman Kodak (30 mg of Stains All, 100 ml of dimethyl formamide, and distilled water to 1000 ml) or by the silver stain technique (28).

X-ray Investigations

The x-ray diffraction patterns were recorded on CEA films (Ceay- erken, Sweden) a t room temperature with CuKa ( X = 1.5418 A) radiation. Experiments were done either with an Elliott GX 13 or Rigaku R200 rotating anode generator. The Elliott generator was operated a t 35 kV and 35 mA with a focal cup of 100 pm. The primary beam was monochromatized and focused in the vertical direction using a quartz monochromator followed by a gold mirror; no colli- mation was used. The Rigaku generator was operated a t 2.4 kilowatts with a focal cup of 200 pm and a graphite monochromatized and collimated primary beam. Pictures were taken using a Nonius preces- sion camera with a crystal to film distance of 100 mm.

Due to the large unit cell and the important solvent content of the crystals, their diffracting power is very low so that long exposure times (8 h or more) were needed to obtain a reasonably still picture.

RESULTS AND DISCUSSION

Crystallization in the Presence zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Ammonium Sulfate-The optimal range conditions for the crystallization of the differ- ent components of the aspartic acid system from yeast in their free and complexed form are summarized in Table I. Both tRNAA"pand the aspartyl-tRNA synthetase can he crys- tallized individually either at low salt conditions (12, 13) or in the presence of ammonium sulfate at 62 and 54% of saturation, respectively, conditions which give the best crystal forms (see Table I). For the complex, however, crystals could only be grown at high salt conditions, between 48 and 53% of saturation of ammonium sulfate, the exact value being related

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Crystals zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof tRNA .Aminoacyl-tRNA Synthetase Complex zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA8431 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBATABLE I

Crystallization conditions of aspartyl-tRNA synthetase, and the complex formed between these two macromolecules in the presence of ammonium sulfate

All numbers refer to starting conditions. The final concentration of ammonium sulfate corresponds to that in the reservoir. For the complex, the final concentration varies with the concentration of the additives. Spermine at concentrations needed for tRNA crystallization is without apparent influence on the crystallization of the complex.

Conditions tRNA"'(Ref. 12) Aspartyl-tRNA synthetase (Ref. 13) Complex ([tRNA]/[enzyme] zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA= 2)

Precipitant (NHI)zSOI (NHI)zSOI (NK)ZSOI Starting concentration 40% 40% 36% Final concentration 62% 54% 48 to 53%

tRNA 3 to zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA5 mg. ml" 1.7 to 2.8 mg.ml-l Enzyme 4 mg. ml-I 3 to zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA12 mg. ml"

Concentration 15 mM 15 mM 15 to 60 mM

Molecular concentration

Buffer Cacodylate (Na) MES (KOH) Tris (HCI)

PH 6.8 6.7 7.8 to 8.5 Additives (nature and concentration) Spermine, 3 mM

MgCIz,l5 mM +Spermine

MgCIz, 0.06 mM +M&Iz, 5 to 15 mM

EDTA, 0.03 mM EDTA, 0.25 mM Dithioerythritol, 0.06 mM Dithioerythritol, 0.25 mM

Temperature ("C) 20 4 4-20

to the concentration of macromolecules, the temperature, and the presence or absence of additives to the crystallization medium.

Other conditions tried, for example, various types of poly- ethylene glycol, methylpentanediol, isopropyl alcohol, etc., as precipitants, were unsuccessful. In ammonium sulfate, an important parameter for crystal growth is the pH range. The best crystals are obtained in a rather alkaline pH range (7.8 to 8.5) compared to the more acidic conditions needed for the crystallization of tRNAAsp and aspartyl-tRNA synthetase alone (pH 6.7 to 6.8). The main additive used is magnesium chloride; it seems to affect slightly the external shape of the crystals but it does not improve their diffracting ability (see below). Most likely, it affects the crystal growth by increasing the solubility of the complex and thus lowering the speed of growth. The resulting crystals are usually well shaped with well formed external faces. However, it must be stressed that crystals that diffract well can be grown without the addition of magnesium chloride, whereas this was not possible with the free components. Addition of spermine (up to 3 mM) as well as the nature of the buffer (Tris, MES, HEPES, and cacodylate) and its concentration, reducing agents (2-mercap- toethanol, dithioerythritol, cysteine, glutathione, even at high concentrations up to 5 mM) are without apparent influence. Crystals can be obtained over a large range of temperature. Big crystals can grow in a few hours a t room temperature, but these are usually very fragile and difficult to handle. Routinely good crystals are prepared at 4 "C in a cold room.

In view of neutron diffraction studies, crystallization at- tempts were conducted in the presence of heavy water. Suc- cessful crystallizations were conducted under the conditions described before with various D20 concentrations (50 to 80%). I t should also be noted that the presence of the nonionic detergent 1-0-octyl-P-D-glucopyranoside at 0.1% does not affect crystal growth.

Some typical crystal habits are shown on Fig. 1. A close-up view of a crystallization drop is presented on Fig. 1A. The crystals are of cubic shape; their sizes range from a few micrometers up to a 0.5-mm edge. Sometimes a few well shaped larger ones are obtained with dimensions up to 0.8 mm (Fig. 1R). Very often large crystals grow either from redissolved small ones or from precipitated material. This could easily be followed by visual inspection; around the growing crystal the boundary of precipitated material or of microcrystals retired. For these large crystals (see Fig. 1B) the external shape is generally not a perfect cube, but is

--;w

FIG. 1. Crystal habits of the tRNAAEp.aspartyl-tRNA syn- thetase complex. A, close-up view of a crystallization drop; zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAR, typical crystal used for x-ray investigations; C, particular crystal form obtained under quick growth conditions. The three pictures are taken from experiments run under similar optimal conditions (see "Mate- rials and Methods"). The scale bars represent 0.25 mm.

determined by the size of the drop. A commonly encountered aspect of the cubic crystals when these grow too quickly is illustrated in Fig. 1C. For example, the deformations which result in concave faces and rounded corners are often observed when crystals are grown at room temperature.

Importance of the Nucleic Acid-Protein Stoichiometry-By far the most important factor in crystal formation appears to be the stoichiometry of the components (n = (tRNAAsp]/ [aspartyl-tRNA synthetase]). The resulting analysis is sum- marized on Table 11. Five conditions were considered. In the absence of tRNAAsp (control experiment), the typical tetrag- onal pyramids characteristic for aspartyl-tRNA synthetase (13) are obtained. No crystals can be grown for values of n up to 1.8. The best crystals are obtained when the mother liquor contains two tRNA molecules for one molecule of enzyme. This ratio corresponds to the saturated dimeric enzyme and cannot be altered by more than 10%. One should stress the point that concentration measurements of macromolecules are done with an accuracy of 10% and that the stoichiometry values given in Table I1 reflect this experimental uncertainty. This practical limitation is overcome by trial and error setups around the theoretical ratio of 2.0. The importance of stoi- chiometry is emphasized by the result of experiments in which

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8432 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBACrystals zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof tRNA .Aminoacyl-tRNA Synthetase Complex zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBATABLE I1

Influence of the rtRNAA*pl//a~partyl-tRNA synthetase] stoichiometry on the crystal growth

All crystallization conditions are similar (see optimal conditions under "Materials and Methods") except for the [tRNA]/[aminoacyl- tRNA synthetase] stoichiometry. It can be noted that the tetragonal crystals of aspartyl-tRNA synthetase are also obtained under complex crystallization conditions. AspRS is for aspartyl-tRNA synthetase. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Ocn<1.8 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAI N o crys ta ls j I / I

crystals were grown by adding the appropriate amount of tRNA in crystallization drops in which the initial tRNA- synthetase ratio was 1.0.

In the presence of an excess of tRNA (2.2 < n < 2.5), the crystal shape can be modified. As illustrated in Table 11, the sharp edges of the cubes become rounded. For a yet higher excess of tRNA (n > 2.5), the cubic morphology of the crystals disappears and is replaced by spherolites which can reach large sizes.

In conclusion, it appears that the best crystals are obtained when the enzyme is perfectly saturated by tRNA and when no excess of free macromolecules is present.

Characterization of tRNAAsp and Aspartyl-tRNA Synthetase from Dissolved Crystals-The putative tRNAAsP and synthe- tase originating from crystals were identified by gel electro- phoresis methods and by biochemical activity assays of both types of macromolecules. All experiments described below were carried on with crystals carefully washed with fresh mother liquor so that the possibility of contamination by noncrystallized material could be eliminated. Furthermore, each series of assays was done with one unique crystal. A typical electrophoresis experiment is displayed on Fig. 2; it clearly shows the presence of two components originating from one dissolved crystal migrating on the gels as control aspartyl-tRNA synthetase (A) and tRNAAxp (23).

The biological activity assays reported in Table 111 permit identification of the tRNA and synthetase nature of the two components shown on the gels. The presence of aspartyl- tRNA synthetase was demonstrated by the aminoacylation of exogenous tRNAAsp added to the medium. Two types of ex- periments allowed the demonstration of the presence of the tRNA. With and without addition of exogenous aspartyl- tRNA synthetase, it was possible to recover an equivalent amount of trichloroacetic acid-precipitable radioactivity, clearly indicating the aspartylation of a significant amount of

The aspartylation assay of the tRNA enabled us to easily calculate the total amount of tRNA in one crystal. As given

tRNA.

in Table 111, a standard size crystal (0.4-mm edges) contains about 5 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBApg of tRNA. The amount of about 12 pg of synthetase from the same crystal was estimated from specific activity measurements of' the enzyme, gel electrophoresis, and crys- tallographic data. The measure based on specific activity implies similar values of this activity for the control and crystallized synthetase. Estimation by gel electrophoresis, the less accurate method, was based on a densitometric compari- son of the crystalline protein with a pre-established scale of control synthetase (data not shown). Finally, the accuracy of the crystallographic method strongly depends upon crystal

Enzyme

s c zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAA

94 -

45-

t R N A

s c s

I) -25 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA30-

FIG. 2. Electrophoretic characterization of aspartyl-tRNA synthetase (A) and tRNA"' (B). The patterns were obtained with material originating from one crystal (C) and with control macro- molecules in solution zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(S). The crystal (-0.4-mm edges) was dissolved in 20 pl of buffer solution, and = 1 pg of enzyme or ~ 0 . 5 pg of tRNA was deposited on the gels. The gels were calibrated for molecular weights (in kilodaltons).

TABLE 111 Transfer RNA aminoacylation assays with material originating from dissolved crystals and estimation of the content of tRNA and enzyme

A crystal of - 0.4-mm edges was dissolved in 20 pl of buffer (see "Materials and Methods"). Aliquots of 1 pl (A) or 3 pI (€3 and C) were used for 100-pl incubation mixtures. For enzyme and tRNA activity measurements, the mixtures were supplemented with 10 pg of tRNAAaP and 1 pg of aspartyl-tRNA synthetase, respectively. In- cubation was for 30 s a t 37 "C and results are expressed in counts/ min for 40 pl of reaction mixture. When tRNA or complex were tested, similar results were obtained after a 5-min incubation. The experimental values indicated in the table give an estimation of 4.2 pg of tRNAAnp(=0.17 nmol) and 9.5 pg of aspartyl-tRNA synthetase (-0.08 nmol) uresent in the crvstal.

Macromolecules Macromolecule ~ ~~

tested added to assay Control Mother Dissolved (in excess) liquor crystal

cpm - "~

A Aspartyl-tRNA tRNAA*p 75 82 20,350

H tRNAA"" Aspartyl-tRNA 81 70 3,282

C Complex 71 85 3,779

synthetase

synthetase

. - . - "~

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Crystals zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof tRNA .Aminoacyl-tRNA Synthetase Complex zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAA - & < & i ; B

hhl (A) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAand hkO (B) reciprocal lat- FIG. 3 . Precession photographs of

tice zones. The precession angle was 3.5' and exposure time was 24 h.

- e zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA''e zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA[ o o zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAI1 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAt, I , zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1" zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

size measurements (see below). Despite the intrinsic limita- tions of the three methods, the results agree within 20%. From these experimental data one can deduce a stoichiometry of two tRNA molecules for one synthetase within the crystal. This stoichiometry is the same as that found in solution (14).

X-ray Data-X-ray precession photographs of hkO and hhl reciprocal lattice zones are shown on Fig. 3. The hkO zone exhibits 4-m symmetry with equal lattice parameters and h + k = 2n existence condition for the spots. The hhl layer shows mm symmetry with lattice parameters equal to 2a and a&. Therefore, the crystals belong to the cubic system. This agrees with the optical properties of the crystals which are inactive under polarized light. The unit cell parameter is 354 A which corresponds to a volume of 4.43 x 10' A'. The systematic absence of hkl for h + k + 1 = 2n + 1 leads to the space group I432 (space group number 211) with 48 asymmetric units/ unit cell.

Crystals are sensitive to x-ray damage. Most of them exhibit considerable weakening of the diffraction pattern after 24-!! exposure. Diffracted intensities can be observed up to 7.5-A resolution with th? best crystals, but routinely, patterns ex- tend out to 8 to 9-A resolution. Crystals grown in the presence of magnesium chloride seem more affected by a loss of reso- lution and radiation damage. This might be due to magne- sium-activated hydrolytic cleavage of the RNA backbone (29). Crystals are sensitive to temperature changes. Increasing the temperature above 20 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA"C led to changes in their physical aspect. The crystals became irreversibly insoluble, opalescent, and mechanically rubber-like.

Due to the size of some of the crystals, a direct estimation of their tRNA content was possible. A typical experiment is shown in Fig. 4. A large crystal (0.8 x 0.8 x 0.4 mm) was dissolved after being measured with a binocular microscope and carefully washed with mother liquor. An UV absorption spectrum was then obtained on both the solution containing the redissolved crystal and the buffer solution used to wash the crystal. The results gave an optical density a t 260 nm consistent with the presence of 22.1 pg of tRNA, assuming that one absorbance unit a t 260 nm equals 40 pg of tRNA/ ml. The volume of an elementary unit cell being equal to 4.43 x 10' A", the resulting tRNA content of one unit cell is then equal to

22.1 x x 6.023 x lo2% x 4.43 x 10'

24,160 x 2% x lo1*

2 96 tRNA molecules/unit cell

where 24,160 is the tRNA molecular weight, 256 X 10'' A:' is the crvstal volume, and 6.023 x lo2:' is the Avogadro number.

b h O O 1

0.75

Ly

V Z 0.50 m U

51 E

rn 4:

0.25 \ * Buffer I 1

220 260 360

8433

Wavelength (nm)

FIG. 4. Ultraviolet absorption spectrum of a dissolved com- plex crystal. The crystal (0.8 X 0.8 X 0.4 mm) shown in the inset was stabilized and washed with mother liquor containing ammonium sulfate at 7 0 6 of saturation. This large crystal was originally assigned to neutron diffraction experiments (at the Institut Laue Langevin, Grenoble, France) but it broke during mounting in a quartz capillary. The crystal was then dissolved in 840 pl of 50 mM Tris-HCI buffer at pH 7.2 and its optical absorption spectrum was recorded in a cuvette of 10-mm pathway.

The result implies the presence of two tRNA molecules in the asymmetric unit. The influence of the enzyme on the absorp- tion a t 260 nm is negligible. If one assumes one molecule of enzyme for two tRNA molecules, as demonstrated in solution (13), the crystal contains 55.2 pg of enzyme, which contributes to the optical density by 0.028 absorbance unit a t 260 nm, i.e. about 3% of the observed value.

Assuming then one molecule of enzyme and two molecules of tRNA/asymmetric unit, the v$ue of crystal volume/unit of macromolecular weight is 5.3 A'/Da, outside the standard range for proteins (1.68 to 3.53) (30). For a partial specific volume of 0.7 cm:'/g, the solvent content of the crystal would then be 78%, a large value consistent with the particular softness of the crystals.

GENERAL DISCUSSION

Although crystals from viruses were among the first biolog- ical material crystallized (tobacco mosaic virus was crystal-

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8434 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBACrystals zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof tRNA .Aminoacyl-tRNA Synthetase Complex zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAlized by Stanley in 1935 (31)), only a few other protein-nucleic acid complexes have up to now been crystallized (31-37). Until recently, the only crystals available, beside viruses (for a review see Ref. 32), were obtained with nucleosome core particles (33). All these particles are multimolecular com- plexes isolated as such in which the interactions between the individual components do not involve sequence-specific nu- cleic acid-protein recognition. Here, the main function of the proteins is to pack or protect the nucleic acid. One of the consequences, in the case of nucleosomes, is that crystals are formed with particles containing heterogeneous DNA se- quences. As for the spherical viruses, in all the structures solved a t high resolution the RNA part is disordered and cannot be seen on the electron density maps (38-40). For the nucleosome core particles a low resolution structure was ob- tained using different approaches, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAi.e. electron microscopy (33), x-ray, and neutron diffraction (41, 42). In that case, the relative positioning of the components and the gross shape of the DNA part are clearly shown. More recently the small ribosomal subunit (34) and the complete ribosome (35) have been crystallized as well as complexes between restriction endonucleases and DNA fragments (36, 37).

The crystals described in this report are made of a tRNA with its cognate aminoacyl-tRNA synthetase. The recognition process between the enzyme and its macromolecular substrate certainly involves specific interactions between complemen- tary structural features. This can result from pre-existent complementarity or from finely tuned adjustment of the com- ponent conformations. Experimental data in solution favor this last hypothesis (e.g. Refs. 1, 43, and 44). Indirect evi- dences for that are the two known three-dimensional struc- tures of aminoacyl-tRNA synthetases which both exhibit local disorders (10, 11). It is tempting to think that the disordered parts are involved in tRNA binding and that their flexibility enables a dynamical binding of the nucleic acid. If that is true these parts of the structure would be fixed to the substrate and then become more ordered in the complex crystals. How- ever, in the yeast aspartic acid system the limited resolution of the actual crystals, lower than that of the free enzyme, prevents any positive conclusion. But one should stress the unusually high solvent content which accounts for a slight disorder in the packiag and likely explains why the resolution does not exceed 7.5 A.

High salt conditions have long been thought to prevent complex formation or at least largely to minimize their sta- bility. This belief relies on the assumption that association between nucleic acids and proteins involves mainly electro- static interactions which are hampered by high ionic strengths. For that reason, most if not all previous attempts to crystallize these complexes have been carried on in low salt conditions (13, 45) using, for example, alcohols or polyethyl- ene glycol as precipitating agents. However, the fact that both individual components of the yeast aspartic acid system crys- tallize using ammonium sulfate a t high concentrations as precipitant was a strong incitation to try similar conditions for the complex. At that point, a paper by Von der Haar (46) describing a tRNA effect on the elution of aminoacyl-tRNA synthetases from Sepharose 4B columns by reverse ammo- nium sulfate gradients pointed to the probable existence of tRNA-protein interactions in the presence of this salt. This was reinforced by the older observations of Thiebe and Zachau (47) and Feldmann and Falter (48) showing significant het- erologous aminoacylation of tRNAs at high ionic strength with various salts. These facts underlined the potentiality of ammonium sulfate as precipitant in crystallization, but more tests were necessary to check the biochemical significance of the complex formed under these unusual conditions. Complex

formation in an ammonium sulfate solution (at 40% of satu- ration) was proven for the yeast aspartic acid system by fluorescence and neutron small angle scattering techniques (14). The aminoacylation reaction could also be carried out in that medium (14). In the presence of other salts at very high concentrations, e.g. NaCl and NH,Cl, no complex could be observed. These solution studies clearly showed the speci- ficity of action of ammonium sulfate compared to other salts, a fact yet not well understood.

CONCLUDING REMARKS

The successful crystallization of the complex formed be- tween tRNAAsp and aspartyl-tRNA synthetase using ammo- nium sulfate at high concentration suggests a possible expla- nation of general significance. High ionic strength would inhibit the formation of nonspecific electrostatic interactions responsible for heterogenous populations (43, 49) and favor hydrophobic interactions and more specific hydrogen bond associations. Two experimental evidences enforce this expla- nation. (i) Addition of a small amount of yeast tRNAAsP to a solution of aspartyl-tRNA synthetase induces the precipita- tion of the enzyme, when done in low ionic strength but otherwise not; and (ii) small angle neutron scattering of complex clearly shows the presence of aggregates which dis- appear after increase of ionic strength (14).

Structural investigations on the crystals described in this paper are underway. Neutron and x-ray diffraction as well as electron microscopy are expected to give a more accurate picture of the structure of tRNA. aminoacyl-tRNA synthetase complexes and to bring a deeper insight on the mechanism of protein-nucleic acid recognition.

Acknowledgments-We are grateful to Dr. D. Kern for stimulating discussions and help during enzyme handling. We acknowledge the contribution of Drs. G. Dirheimer, J. Gangloff, and G . Keith who first purified and studied the components of the aspartic acid system. We thank M. Schlegel for help with countercurrent distribution of crude tRNA. Thanks are also due to Dr. B. Jacrot for providing us with experimental facilities at the European Molecular Biology Lab- oratory outstation of Grenoble and for constant support and encour- agement.

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