PROTEINS Structure, Function, and Genetics 24516-519 (1996)

Purification, Crystallization, and Preliminary X-Ray Diffraction Studies of tRNA-Guanine Transglycosylase From Zymomonus mobilis Christophe Romier,' Ralf Ficner,' Klaus Reuter? and Dietrich Suck' 'European Molecular Biology Laboratory, Structural Biology Programme, 691 17 Heidelberg, Germany; 2Znstitut fur Biochemie, Universitat Erlangen-Nurnberg, 91 054 Erlangen, Germany

ABSTRACT The tRNA modifying enzyme tRNA-guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-ami- nomethyl-7-deazaguanine. Tgt from Zymomo- na8 mobilis has been purified by crystallization and further recrystallized to obtain single crys- tals suitable for X-ray diffraction studies. Crys- tals were grown by vapor diffusionlgel crystal- lization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 A, b = 65.1 A, c = 71.9 A, and p = 97.5", and contain one molecule per asym- metric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 A resolution. o 19% Wiley-Liss, Inc.

Key words: tRNA, transglycosylation, queu- osine, crystallization, X-ray dif- fraction

INTRODUCTION Modified nucleosides are a characteristic feature

of RNAs. Up to 93 modified nucleosides have been reported so far.' In the single class of tRNAs, 79 have been found. Although in many cases their pre- cise function is not known, it appears that they lead to optimization of tRNA function, and are used as a regulatory device in cellular processes and as a sen- sor of the metabolic status of the

Among the modified tRNA nucleosides, queuosine [Q:7-(((4,5-cis-dihydroxy-2-cyclopentene-l-yl)amino)- methyl)-7-deazaguanosine] is one of the bulkiest (Fig. 1A). It is present in the first position of the anticodon of the tRNAs specific for Tyr, His, Asp, and Asn in most eucaryotes and e~bac ter ia .~ The exact function of Q in tRNAs is not yet clarified, but it seems to participate in the fine tuning of protein biosynthesis as it is found for other tRNA modifica- t i o m 2 There is evidence for a distinct function of Q in eubacterial tRNAs where a mutation in a Q-bio- synthesis gene causes apathogenicity in the dysen- tery bacterium Shigella f l e ~ n e r i . ~ In eucaryotes, the Q-base is a nutritient5 and appears to play a role in

0 1996 WILEY-LISS, INC.

different cellular events such as differentiation, pro- liferation, and ~ i g n a l i n g . ~ , ~ Q-undermodified tRNAs might be required for frameshifting of some retrovi- ruses.7

Q-base is synthetized de novo in eubacteria by several enzymatic steps, not all of them being yet known.8 Only two of the enzymes involved have been characterized and their corresponding genes cloned: the tRNA-guanine transglycosylase encoded by the tgt gene and the S-adenosy1methionine:tRNA ribosyl transferase-isomerase encoded by the queA gene. The tgt and queA genes were found in one operon in Escherichia ~ o l i . ~ The role of Tgt is to ex- change the genetically encoded guanine at the wob- ble position with the queuine precursor preQl (Fig. l)."," Recent studies have demonstrated that a minihelix containing the anticodon stem and loop region of tRNATyr is a substrate for the enzyme" and that a YUGU (Y = pyrimidine) sequence in the anticodon loop is the only requirement for this mini- helix to be efficiently recognized by Tgt. This se- quence corresponds to bases 32 to 35, base 34 being the modified guanine.13

Recently, the tgt gene from Zymomonas mobilis was c10ned.l~ The E . coli and Z . mobilis tgt genes have been overexpressed in E. coli and their prod- ucts purified to h~mogene i ty . ' ~ ,~~ Although both en- zymes show more than 60% identity, the Z . mobilis Tgt is a monomer whereas the one from E. coli tends to form t r i m e r ~ . ' ~ , ~ ~ E . coli Tgt contains one zinc ion and the residues serine 90 and cysteine 265 are re- quired for enzymatic activity. 16,17 The zinc ligands, serine 90 and cysteine 265 are all conserved in the Tgt of Z. rn0bi1is.l~

We report here an improved purification protocol

Abbreviations: Tgt, tRNA-guanine transglycosylase; Q, queuosine; Q-base, queuine; PEG, polyethyleneglycol; SDS- PAGE, sodium dodecylsulfate polyacrylamide gel electro- phoresis; DTT, dithiothreitol; EDTA, ethylene diamine tet- raacetic acid; DMSO, dimethylsulfoxide; pOG, n-OCtyl-p-D- glucopyranoside; MPD, 2-methyl-2,4-pentandiol

Received October 17, 1995; accepted October 27, 1995. Address reprint requests to Dietrich Suck, European Molec-

ular Biology Laboratory, Structural Biology Programme, Mey- erhofstrasse 1, 69117 Heidelberg, Germany.

CRYSTALLIZATION OF Tgt 517 A.

NHz I

Guanine (G) preQueuine1 (preQ,) Queuine (Q-base)

Fig. 1. A: Structures of guanine, preQ, and Q-base. B: Reaction catalyzed by Tgt: exchange of guanine with preQ, on the tRNA.

of the Tgt from 2. mobilis, the technique we em- ployed to obtain crystals suitable for X-ray diffrac- tion studies, and the results of a preliminary crys- tallographic analysis of these crystals.

MATERIALS AND METHODS Purification

Cloning, overexpression in E. coli, purification and characterization of 2. mobilis Tgt have been previously reported.', We further modified the pu- rification protocol, as described below, to take ad- vantage of the enzyme's crystallization tendencies in the presence of low salt. All purification steps were carried out at 4°C.

The cell pellet from a 3 1 culture (approx. 10 g of cells) was resuspended in 50 ml of buffer A (10 mM Tris/HCl, pH 7.8; 10 mM MgC1,; 1mM DTT; 1 mM EDTA) plus 0.2% Nonidet P-40 (Sigma, Deisenho- fen, Germany) and 1 mM Pefabloc (Boehringer, Mannheim, Germany). Cells were disrupted by ad- dition of lysozyme (Sigma) to a final concentration of 1 mg/ml, and by sonication (15 cycles of 20 sec od45 sec off). The cell extract was subjected to a first cen- trifugation at 12,OOOg for half an hour. The super- natant was further submitted to a second centrifu- gation at 100,OOOg for 2 hours.

The supernatant of the second centrifugation was loaded onto a Q-Sepharose (Pharmacia, Uppsala, Sweden) column (bed volume: 53 ml) and, after washing with buffer A, the enzyme was eluted with a linear 0 to 0.3 M NaCl gradient (300 ml) in the same buffer with a flow rate of 1.5 ml/min. The Tgt eluted at 0.2 M NaC1. (NH,),SO, was added to the Tgt containing fractions to a concentration of 0.5 M. Subsequently, these fractions were loaded onto a HiLoad Phenyl-Sepharose (16/10) column (Pharma- cia). The enzyme was eluted by washing the column

with buffer A plus 0.5 M (NH,),SO,, leading to a purity (estimated by SDS-PAGE) of 80-90% de- pending on the amount of protein loaded. The Tgt containing fractions were then pooled and concen- trated to reach a protein concentration of 10 mg/ml (estimated with the Bradford method"). The con- centrated fractions were dialyzed (cutoff 12,000- 14,000) overnight against buffer A. During dialysis, the Tgt precipitated in form of microcrystals. The microcrystals were washed several times with buffer A to remove any protein contaminant.

Crystallization The microcrystals obtained by dialysis were too

small for X-ray diffraction experiments. These pro- tein crystals were redissolved in a high salt buffer (10 mM HEPES, pH 7.5; 2 M NaC1; 1 mM DTT). The volume of the added buffer was adjusted to reach a protein concentration of 12 mg/ml.

Crystallization experiments were performed by the hanging drop vapor diffusion method at 25°C using Linbro 24-well culture plates. The protein sample was mixed (2 pl + 2 p1) with the well solu- tion (100 mM Tris/HCl, pH 8.5; 13% PEG 8,000; 1 mM DTT) containing agarose for gel electrophoresis (Life Technologies, Paisley, Scotland) and equili- brated over 0.5 ml of well solution. The agarose/PEG solution was prepared by mixing the well solution, kept a t 37"C, and a 1% agarose solution, kept at 95"C, to have a final agarose concentration of 0.2%. This solution was subsequently used for mixing with the protein solution. Crystals appeared in the course of 2 days and grew over a few weeks to reach a max- imal size of 0.2 x 0.2 x 0.05 mm3. Single crystals were then transferred to a fresh droplet made of 2 p1 of protein solution and 2 p1 of well solution (contain- ing only 5% PEG 8,000), and equilibrated over 0.5

518 C. ROMIER I ET AL.

diffraction experiments. Therefore, we had to redis- solve them in a high salt buffer and recrystallize the protein. For this purpose, we used a factorial screen2' which showed that Tgt crystallized in pres- ence of PEG 6,000, PEG 8,000 and PEG 20,000 as precipitating agents. Under these conditions, we also observed a salting in effect: due to the salt con- centration in the protein solution, the drop in- creased in volume in the course of time, thereby de- creasing the salt concentration. Among the different PEGS used, PEG 8,000 provided the best results.

Tgt crystallized in a pH range of 6.0 to 9.5 at room temperature. However crystals showed a strong ten- dency to cluster, especially at low pH. This tendency decreased with increasing pH, but the majority of the crystals still showed clustering even at pH 9.5. On the other hand, bigger crystals were obtained around pH 6.5. After unsuccessful trials to prevent clustering with various additives (DMSO, Dioxan, POG, MPD), we switched to pH 8.5 where single crystals were obtained more easily, although smaller (maximum size 0.15 x 0.15 x 0.03 mm3) and therefore hardly suitable for X-ray diffraction measurements.

We tried to improve the size of these crystals with different methods. Raising the crystallization tem- perature at 37°C gave bigger crystals but they were embedded in a protein skin, preventing any han- dling. Crystallization in presence of 0.2% agarose21 provided good results, both in terms of clustering reduction and size increase. Still, the size of these crystals remained small (maximum size 0.2 x 0.2 x 0.05 mm3). We, therefore, employed macroseeding techniques22 to make them grow bigger. Crystals were transferred to a fresh drop having no agarose and a lowered PEG concentration (5% rather than 13%). First the crystals began to dissolve due to the higher salt concentration in the drop. The equilibra- tion of the drop and the decrease in salt concentra- tion, however, rapidly prompted the crystals to grow again and reach a size of up to 0.4 x 0.4 x 0.1 mm3 after a few weeks. It should be noted that crystals grown without agarose could also be used for mac- roseeding. However, they never reached more than 0.07 mm in the third dimension.

Crystal Characterization Native data sets were first collected at 4°C on a

MAR imaging system (18 cm) equipped with a Mac- Science MX18 generator (Siemens, Karlsruhe, Ger- many) operating at 40 kV and 90 mA. Crystals showed diffraction to beyond 2.0 A. After processing the data with XDS, the space group was unambigu- ously determined as C2 (monoclinic). However, due to the low symmetry and the decay of the crystals in the X-ray beam, a complete data set could not be collected.

We were able to collect a complete data set at 15°C at the synchrotron in Lure due to the reduced X-ray

TABLE I. Data Statistics*

Completeness (%)

Resolution All Rsym (A) data 1>3a(I) (shell) (%) Reflections 25.0-4.0 4.0-3.2 3 2-2.8 2.8-2.55 2.55-2.4 2.4-2.25 2.25-2.15 2.15-2.05

1.95-1.85 2.05-1.95

93.8 90.9 96.1 94.6 96.1 92.8 95.7 90.4 95.5 89.7 95.1 86.5 94.6 84.5 93.6 80.1 92.7 75.3 84.7 59.8

1.8 1.9 2.6 3.3 3.9 4.3 5.6 6.5 8.8

13.0

8,174 8,140 8,300 8,175 6,674 8,489 7,001 8,098 9,567 9,478

Total number of reflections: 82,096 Unique reflections: 33,727 Overall R,,, (25.0-1.85 A): 2.9% *R,, = ZII-<I>IEI, where I is the intensity for an observa- tion of a multiply observed reflection.

ml of this new well solution. The crystals first began to dissolve but were soon growing again to reach a maximum size of 0.4 x 0.4 x 0.1 mm3 after a few weeks.

Data Collection and Reduction Crystals were mounted in quartz capillaries of 0.5

mm diameter. A complete native data set could be collected at the Lure synchrotron a t a temperature of 15°C using a MAR imaging plate system. The crystal- to-detector distance was adjusted to 280 mm (wave- length of 0.9 & and 1" frames were collected with a 2 min. exposure time. In total, 140 degrees were col- lected. The data set was processed with XDS."

RESULTS AND DISCUSSION

Purification In a preliminary purification protocol of 2. mobilis

Tgt, a gel filtration was used as a final step.14 As we concentrated the Tgt containing fractions after the gel filtration for crystallization purposes, the en- zyme precipitated. The precipitate was analyzed by electron microscopy and showed tiny single micro- crystals. Since the enzyme was soluble at high con- centration in presence of 0.5 M ammonium sulfate, we concluded that the salt decrease was the main crystallization factor. During our subsequent purifi- cation trials, the gel filtration step was skipped and replaced by a dialysis step even though the enzyme was far from purity. This protocol worked very well indeed leading to an extremely pure protein with very high yield.

Crystallization During the purification, we observed that Tgt

crystallized at low salt concentrations. However, these crystals were too small to be used in X-ray

CRYSTALLIZATION OF Tgt 519

damage at a wavelength of 0.9 A. Data extended to a resolution of 1.85 A with a Rsym on all data of 2.9% (Table I). The space group was C2 with cell constants a=92.1 A, b=65.1 A, c=71.9 A, and p=97.5”. As- suming one molecule in the asymmetric unit, the crystal volume per protein mass (Vm) amounted to 2.5 A31Da, corresponding to a solvent content of 51%. This is within the range commonly observed for protein crystal^.'^ Heavy atom search is in progress.

ACKNOWLEDGMENTS We thank T. PrangB, M. Schiltz, and T. Ceska for

the data collection in Lure. We are also grateful to M. Auer who carried out the electron microscopy experiments. This work was supported by a grant of the Deutsche Forschungsgemeinschaft.

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