conformational movements and cooperativity upon amino acid, atp and trna binding in threonyl-trna...

Conformational Movements and Cooperativityupon Amino Acid, ATP and tRNA Binding inThreonyl-tRNA Synthetase

Alfredo Torres-Larios, Rajan Sankaranarayanan, Bernard ReesAnne-Catherine Dock-Bregeon and Dino Moras*

Laboratoire de Biologie etGenomique StructuralesIGBMC, 1 rue Laurent FriesBP 10142, 67400 Illkirch CedexFrance

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylo-coccus aureus, with ATP and an analogue of threonyl adenylate, aredescribed. Together with the previously determined structures of Escheri-chia coli ThrRS with different substrates, they allow a comprehensiveanalysis of the effect of binding of all the substrates: threonine, ATP andtRNA. The tRNA, by inserting its acceptor arm between the N-terminaldomain and the catalytic domain, causes a large rotation of the former.Within the catalytic domain, four regions surrounding the active site dis-play significant conformational changes upon binding of the different sub-strates. The binding of threonine induces the movement of as much as 50consecutive amino acid residues. The binding of ATP triggers a displace-ment, as large as 8 A at some Ca positions, of a strand-loop-strand regionof the core b-sheet. Two other regions move in a cooperative way uponbinding of threonine or ATP: the motif 2 loop, which plays an essentialrole in the first step of the aminoacylation reaction, and the orderingloop, which closes on the active site cavity when the substrates are inplace. The tRNA interacts with all four mobile regions, several residuesinitially bound to threonine or ATP switching to a position in which theycan contact the tRNA. Three such conformational switches could be ident-ified, each of them in a different mobile region. The structural analysissuggests that, while the small substrates can bind in any order, theymust be in place before productive tRNA binding can occur.

q 2003 Elsevier Ltd. All rights reserved

Keywords: threonyl-tRNA synthetase; tRNA; threonine; ATP; conformation*Corresponding author

Introduction

One of the crucial steps in the translation processis the attachment of an amino acid to its cognatetransfer RNA (tRNA). The accuracy of the reactionrelies on aminoacyl-tRNA synthetases (aaRSs),which carry out this catalysis in a two-step reac-tion. The first step involves the recognition of the

cognate amino acid and ATP and the formation ofa reactive aminoacyl-adenylate intermediate. Inthe second step, the amino acid moiety is trans-ferred to the 20 or 30 ribose hydroxyl group of theterminal adenosine of the cognate tRNA.1,2

Although the same basic reaction is performed byall aaRSs, sequence alignments and differences inthe active site structure have led to a partition intotwo classes.3,4 Class II, to which ThrRS belongs, isbuilt on an antiparallel b-fold core, which containsthe active site, and characterized by three signaturemotifs. Each class is further subdivided into threesubgroups, which differ mainly in additionaldomains. The role of these domains is mostly forthe recognition of tRNA determinants. In the case ofThrRS, one domain possesses an editing functionthat enables hydrolysis of mischarged tRNAThr.5

The synthetases must recognize and bind specifi-cally their three ligands: tRNA, ATP and amino

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved

Present addresses: A. Torres-Larios, Department ofBiochemistry, Molecular Biology and Cell Biology,Northwestern University, Evantson, IL 60208, USA;R. Sankaranarayanan, Centre for Cellular and MolecularBiology, Uppal Road, Hyderabad 500007, India.

E-mail address of the corresponding author:[email protected]

Abbreviations used: ThrRS, threonyl-tRNA synthetase;DN-ThrRS, deletion mutant (residues 242–642) of ThrRS;ThrAMS, 50-O-(N-(L-threonyl)-sulfamoyl)adenosine.

doi:10.1016/S0022-2836(03)00719-8 J. Mol. Biol. (2003) 331, 201–211

acid. To achieve this goal, they possess recognitionsites whose stereochemistry and charge configur-ation are complementary to those of the substrates.After an initial understanding of the aminoacyla-tion reaction that was essentially based upon thelock-and-key model, conformational rearrange-ments due to the binding of the substrates havebeen observed in a number of tRNA synthetasesof both classes. In class II, the variety of amplitudesobserved for these movements, small in the case ofAspRS,6 larger in HisRS7,8 and ProRS,8 may reflectvarious idiosyncrastic combinations (each synthe-tase having its own character), sub-class specificity(class IIa as opposed to class IIb), or substrate-specific effect. Until recently, a detailed descriptionof all the different states of a single system couldnot be provided, mainly because of the lack ofdata corresponding to the complex with the cog-nate tRNA bound in the active site.

Several crystal structures of ThrRS with theirdifferent substrates are now available, and arelisted in Table 1. However, a detailed study of theeffect of each of these substrates has not beenmade so far. Most of the structures were obtainedfrom a truncated form of Escherichia coli ThrRS, inwhich the N-terminal domain is missing. Thistruncated synthetase, which will be designated asDN-ThrRS, is fully active in the first step of theaminoacylation reaction. One crystal form of thisenzyme shows simultaneously the threonine-bound as well as the unliganded state, as the cog-nate amino acid was found in the active site ofonly one subunit of the dimer.9 The wild-typeE. coli enzyme could be crystallized only in the pre-sence of the cognate tRNA,10 but a detailed under-standing of the effect of tRNA binding could notbe ascertained earlier, due to the absence of thecomplete apo enzyme structure. In this work, twocrystal structures of the closely related prokaryoticenzyme from Staphylococcus aureus (SaThrRS) fillthis gap. One is the structure of the enzyme com-plexed with ATP and threonine, representing asituation before the first step of the aminoacylationreaction, and the other that of the complex with anon-hydrolysable analogue of threonyl adenylate,threonyl sulfamoyl adenosine (ThrAMS), showingthe situation after completion of the first step.From the extensive homology of the E. coli andS. aureus enzymes (57% similarity, 44% identity),very similar three-dimensional structures of the

two enzymes are expected when they are boundto the same ligands. Indeed, both enzymes amino-acylate efficiently the same E. coli tRNA (seeMaterials and Methods, data not shown). On thewhole, this collection of free and complexed statesof ThrRS provides a picture of a class IIa synthe-tase with all of its substrates. The comparison ofall these complexes allows a detailed analysis ofthe effect of each substrate on the conformation ofthe enzyme. Residues that recognize a specific sub-strate in each step of the reaction are identified,and the role of the different regions that form theactive site, their movements and interrelationsduring the aminoacylation reaction are highlighted.

Results

Overall flexibility of the ThrRS structure

The overall structure of SaThrRS is the same asthat of EcThrRS.10 Each monomer of the homodi-mer is made of distinct domains (Figure 1): the cat-alytic domain characteristic of class II synthetases,the C-terminal anticodon-binding domain specificfor subclass IIa, and the ThrRS specific N-terminalextension made of domains N1 and N2, whichwas shown to interact with the tRNA acceptorarm and to be responsible for the editing activity.5

The N2 domain is also found in AlaRS, anotherclass II editing enzyme.5,11 After removal of exter-nal loops, each domain of SaThrRS superimposeswell with its counterpart of EcThrRS, with an rmsdeviation of the a carbons never larger than 1 A(Table 2). This confirms the structural similarity ofThrRS from the two prokaryotic organisms,already inferred from the sequence homology.However, this superposition also illustrates theflexibility of the enzyme, particularly at the levelof the connecting helix which makes the linkbetween the catalytic domain and the N-terminalextension: once the cores of the catalytic domainsof EcThrRS:tRNA and SaThrRS:ATP are superim-posed, an additional rotation of 218 in monomer Aand of 278 in monomer B is required to superim-pose the N2 domains (Figure 1 and Table 2). Thecorresponding rotation of the anticodon-bindingdomain is three to four times smaller. The confor-mational flexibility of this domain is probablyrestrained by its closer contacts with the catalytic

Table 1. ThrRS crystal structures

Synthetase Ligands Monomers/asym. unit Resolution (A) PDB code Reference

E. coli ThrRS tRNA þ AMP 1 2.9 1QF6 10E. coli DN-ThrRS Site A: none 2 2.0 1EVK 9

Site B: threonineE. coli DN-ThrRS ThrAMS 4 1.55 1EVL 9E. coli DN-ThrRS SerAMS 2 1.65 1FYF 5E. coli DN-ThrRS Fragment of thrS operator 8 3.5 1KOG 26S. aureusThrRS ATP þ threonine 2 2.8 1NYR This workS. aureus ThrRS ThrAMS 2 3.2 1NYQ This work

202 Threonyl tRNA Synthetase and its Substrates

domain, in particular at the level of the hydro-phobic patch formed by the system-conserved resi-dues Leu277, Trp536 and Leu537†.

The observed change in orientation of the N-terminal extension may have two causes: the bind-ing of tRNA or the constraints due to the crystalpacking. Since the packing interactions are differ-ent for the two monomers of SaThrRS in the crys-tal, the magnitude of the crystal packing effect canbe roughly estimated from the additional rotationrequired to superimpose the N2 domains in thesame dimer, after superposition of the active sites(Table 2). This rotation (108) is much smaller thanthe rotation observed in the presence of the tRNA,which is therefore likely to be the largest contribu-tor to the observed change of orientation. In order

to allow clamping of the tRNA acceptor armbetween the active site domain and the N2 domain,the space between them needs to open. In theabsence of tRNA, the two domains come closer,and form a more compact structure (Figure 1).

Comparison of the active sites

Apart from these global movements, no majorconformational changes could be detected withinthe anticodon-binding domain or the N-terminalextension (possible changes of the latter duringthe editing process are currently under investi-gation). Therefore, we will from now on focus ouranalysis on the catalytic domain, where importantsubstrate-induced conformational changes areobserved. Figure 2 shows plots of the rms devi-ations of the Ca atoms after superposition of thecatalytic core of ThrRS complexed with its different

Figure 1. Stereo view of the monomers of SaThrRS (green) and the EcThrRS-tRNA complex (blue). The catalyticdomains are superimposed. The N-terminal domain in the complex is tilted by more than 208 relative to the freesynthetase, the anticodon-binding domain by about 78. The axes of the two rotations are indicated. This Figure hasbeen made with the program MOLSCRIPT.34

Table 2. Domain superpositions on the SaThrRS:ATP structure

Displaced molecule Target of the superposition Domain Drms (A) k (deg.) D (A)

EcThrRS:tRNA SaThrRS:ATP monomerA Active site 0.7 – –N2 0.8 20.9 6.2C term 0.8 6.4 1.5

EcThrRS:tRNA SaThrRS:ATP monomerB Active site 0.8 – –N2 1.1 27.0 10.8C term 0.7 7.4 1.8

SaThrRS:ATP monomer B SaThrRS:ATP monomer A Active site 0.8 – –N2 1.0 10.0 4.9C term 0.4 1.9 0.4

k is the angle of the additional rotation required to superimpose the N2 N-terminal domain or the anticodon-binding C-terminaldomain, once the active site domain is superimposed on that of SaThrRS. D is the corresponding displacement of the center of massof the domain. Drms is the rms deviation of the superimposed Ca atoms. The tRNA in the E. coli structure induces a large rotationof the N2 domain. The last three lines show the results of the superposition of the two active sites within the same homodimer, andillustrate the effect of asymmetric packing forces in the crystal. The superpositions were made on the core of each domain, obtainedafter removal of the loops containing insertions or deletions in the sequence alignment of E. coli and S. aureus ThrRS, and of someregions of high mobility like the four mobile regions of the active site domain discussed in the text. The least-squares superpositionsinvolve 138 Ca atoms in the active site domain, 84 Ca atoms in domain N2 and 54 Ca atoms in the C-terminal domain.

† Throughout this article the ThrRS aminoacidnumbering will be that of E. coli.

Threonyl tRNA Synthetase and its Substrates 203

substrates. Four regions of the catalytic domainmove significantly upon binding of the substrates.They surround the active site and tend to close onit upon ligand binding, as seen in Figure 3. Thefirst region in the sequence includes residues 301–317 and is located in the zone corresponding to

the so-called ordering loop in ProRS and HisRS,8

which is subjected to order/disorder transitionsfollowing substrate binding. This is topologicallyequivalent to the flipping loop in AspRS,6,12 whichswitches between “open” and “closed” confor-mations according to the status of the reaction.The second region, the motif 2 loop (residues 363–377), connects the two strands of the active-site b-sheet forming the signature motif 2. A movementof this region upon ATP binding was described inSerRS,13 HisRS,14 GlyRS,15 ProRS,8 AspRS,6 LysRS16

and AsnRS.17 The third region comprising residues417–466 is located between the signature motifs 2and 3 and is equivalent to the HisA or histidine-1loop in HisRS7,8 and the proline loop in ProRS.8

The conformation of this loop depending mainlyon the presence or absence of the amino acid, wewill call it the threonine loop. The fourth regionincludes residues 468–480, and has been reportedas a poorly ordered loop in HisRS.7,8 It will bedenoted here as ATP loop because its conformationis mainly dependent on the presence of ATP oradenylate. The effects of each substrate on thesefour regions will now be detailed.

Threonine-induced movements

An essential characteristic of ThrRS is the pre-sence in its active site of a zinc ion which bindssimultaneously the a-amino group and the side-chain hydroxyl of the threonine substrate, and

Figure 2. Conformational changes in the active siteupon binding of the substrates: rms deviation of the Ca

atoms after superposition of the active sites. (a) Threo-nine (EcDN-ThrRS:threonine structure versus apo enzyme(the two subunits of the same high resolution structure)).(b) ATP (SaThrRS:ATP:threonine versus EcDN-ThrRS:threonine). (c) Threonyl adenylate (EcDN-ThrRS:ThrAMS versus EcDN-ThrRS: threonine, emptysubunit). (d) tRNA (EcThRS:tRNA:AMP versus EcDN-ThrRS:ThrAMS). See Table 1 for a list of the crystal struc-tures. The binding of the threonine substrate brings con-formational changes in the ordering loop (noted 1), themotif 2 loop (2) and the threonine loop (3), the bindingof ATP mainly in the ATP loop (4). The binding of aden-ylate combines both effects.

Figure 3. A color-coded representation of the mobileregions in the catalytic domain. The apo structure is ingray. The substrates threonine and ATP are representedin the active site. The mobile regions are: 1, the orderingloop, residues 301–317, in yellow as in the EcThrRS þtRNA structure; 2, the motif 2 loop, residues 363–377, inmagenta as in EcDN-ThrRS þ ThrAMS; 3, the threonineloop, residues 417–466, in red as in EcDN-ThrRS þthreonine; 4, the ATP loop, residues 468–480, in greenas in SaThrRS þ ATP. This Figure and the followingones have been prepared with the program DINO(http://www.dino3d.org).


http://www.dino3d.org

discriminates this amino acid against the isostericvaline.9 The protein ligands that coordinate thision (Cys334, His385, His511) are essentiallyunmoved when the amino acid binds. Among the

residues in direct contact with the amino acid, sev-eral do not move or undergo only side-chain con-formational changes. There are, however, tworesidues for which an important movement is

Figure 4. Binding of the substrates, represented with the same color code as in Figure 3 (stereo views). The apo struc-ture is represented in gray. (a) Amino acid recognition. Tyr462 (threonine loop) and Arg363 (motif 2 loop) are shown inthe apo conformation and in the threonine-bound state, as seen in the two subunits, respectively, of the EcDN-ThrRSstructure. (b) ATP recognition. Three residues of the motif 2 loop (in purple), Arg363, Glu365 and Arg375, interactwith ATP. Lys465 seems to play a catalytic role similar to that played by a Mg2þ in most class II synthetase structures.The ATP loop is locked by a salt bridge (Arg476· · ·Glu527) in the apo structure. In the ATP-bound conformation, thissalt bridge is broken and the ATP loop (in green) closes on the active site. (c) tRNA acceptor arm recognition. Residuesfrom all four mobile loops interact with the tRNA at this level. Asn324 and Arg325 near the ordering loop are in con-tact with the tRNA of the other subunit.


observed: Tyr462 and Arg363, which belongrespectively to the threonine loop and the motif 2loop (Figure 4(a)). The conformational change ofthe threonine loop is quite large. It produces therearrangement of a polypeptide chain of 50 resi-dues and may be described as a 148 rotation of thewhole region (Figure 3). This results in a 3 A dis-placement of Tyr462 to a final position at hydrogenbonding distance of the threonine a-amino group(Figure 4(a)). The conformational change of thethreonine loop brings it closer to the 301–317region, the “ordering loop”†, the position of whichit stabilizes by additional interactions, in particularthe stacking of the highly conserved Phe461 onAsn312. Even though there are no direct contactsbetween the threonine substrate and the orderingloop, the stabilization of this region is clearly seenin the structure of DN-ThrRS:threonine, where thethreonine ligand is present in one subunit only.Without being completely disordered as in thecase of ProRS and HisRS,8 the ordering loop exhi-bits much higher B-factors in the empty subunit,up to 75 A2 for the main chain, compared to amaximum of 42 A2 in the monomer containing theamino acid.

In the motif 2 loop, the displacement of the mainchain allows the contact of the guanidinium groupof the class II conserved Arg363 with the carboxy-late moiety of the threonine. The rearrangement ofthis loop has previously been correlated with thebinding of ATP.13,18 Its conformational dependenceon the amino acid has been noted only in theLysRS system, a synthetase of class IIb.19 In thecase of ThrRS, this conformational change, togetherwith that of the threonine loop, leads to an import-ant reshaping of the active site cavity upon threo-nine binding.

A similar conformational change of the threo-nine loop and of the motif 2 loop is observed inthe structures containing the adenylate analogue,indicating that they are essentially independent ofthe state of the threonine substrate, free or boundto AMP.

ATP-induced movements

In E. coli, as in all structures of class II synthe-tases with ATP or adenylate, the adenine ring isstacked between an invariant arginine of motif 3(Arg520 in EcThrRS) and a highly class II-con-served phenylalanine (Phe379). One peculiarity ofSaThrRS is that the phenylalanine is replaced by amethionine. All other residues involved in ATPrecognition belong to one of the mobile regions.The ATP adopts the class II-specific bent confor-mation with the g-phosphate pointing towards theadenine ring (Figures 4(b) and 5(a)).20 This confor-

mation, however, is seen in one subunit of SaThrRSonly. In that subunit, several class-conserved resi-dues of the motif 2 loop (Arg363, Glu365, Arg375)play a role in the stabilization of the phosphates.The a-phosphate is contacted by Arg363, which,as mentioned, binds also the threonine substrate.A Mg2þ bridges the b and the g-phosphates. Asin other class II synthetases, this magnesium ioncoordinated by the conserved glutamate Glu365, isimportant for the stabilization of the pyropho-sphate. The g-phosphate is further stabilized byArg375, which is a basic residue in all class-IIsynthetases (it is an arginine in class IIa and inmost cases a histidine in class IIb). Arg375 andGlu365 form a salt bridge, which seems essentialfor anchoring the pyrophosphate in the correctposition. In SaThrRS, Arg375 is engaged in asecond salt bridge with Glu484, but this is not con-served (Figure 5(a)). The stabilization of the a andb phosphates involves also the system-conservedLys465, which belongs to the threonine loop. TheLys465 side-chain is displaced by 3 A due to arearrangement of the threonine loop (Figure 4(b)).Lys465 is topologically equivalent to Arg259 inHisRS, which was shown to replace the catalyticMg2þ bound to the a-phosphate in most class IIstructures.2,21 It may play a similar catalytic role,by increasing the electropositivity of the a-phos-phate and thus the stability of the intermediatestate during the first step of the reaction.

Upon binding of ATP, the major movement isthat of residues 468–480, constituting the ATPloop, where the Ca chain is displaced by as muchas 8 A. These residues are located at the level of ahairpin motif involving two adjacent b-strands ofthe core b-sheet. The same displacement is seen inthe structure complexed with ThrAMS, the ana-logue of threonyl adenylate, and in theEcThrRS:tRNA:AMP complex and seems to be trig-gered mainly by the ribose 20 and 30 hydroxyls.These hydrogen-bond the main-chain oxygenatoms at positions 479 and 480 of the ATP loop(Figure 4(b)). In the apo and threonine bound state,the loop is locked by a salt bridge between Arg476and Glu527: this is broken in the presence of ATPor adenylate. This salt bridge is system-conserved,which suggests either that stabilization is requiredin the absence of ATP or adenylate, or that thebreakage of the salt bridge is an important signalfor the reaction.

In the second subunit of the crystal structure, theATP molecule is in a significantly different confor-mation (Figure 5(b)). It is more extended, but notas completely as in class I synthetases.22 The con-formational change can be described as a rotationof about 458 of the pyrophosphate PbPg around theO50–Pa bond. This conformation is stabilized in away different from the first subunit. Here, the gphosphate is bound by Arg363 while Arg375 is dis-placed by about 1 A away from the active site dueto a movement of the motif 2 loop caused by adifferent packing environment. The salt bridgesmade by this arginine in the first subunit are

† Strictly speaking, in ThrRS this region is not a loop inthe usual conformational meaning. However, for thesake of comparison with other studies, we will maintainthis denomination.


much weaker or non-existent, with N· · ·O dis-tances increased by about 0.5 A. Glu365 is stillat hydrogen bonding distance of the g-phosphatebut far from the b-phosphate and unable to binda Mg2þ bridging the two phosphates, which isconsistent with the absence of any significantresidual electron density attributable to such anion. Moreover, Lys465, for which we have postu-lated a catalytic role in the first subunit, is atmore than 4.5 A from any atom of the extendedATP. For these reasons, this conformation of theATP molecule is probably not active. It may rep-resent an intermediate state of ATP as it entersor leaves the active site.

In both subunits of the SaThrRS:ATP structure, asignificant residual density is seen at the threoninesite (Figure 5(a) and (b)). The position of this den-sity, and its shape similar to the threonine moietyin the adenylate analogue (Figure 5(c) and (d)),strongly suggest the presence of threonine mol-ecules together with ATP in the active site, eventhough no threonine was included in the crystalli-zation solution. The presence of threonine isfurther confirmed by the observation that all ofthe threonine-triggered displacements describedabove, in particular that of Tyr462, are seen in theSaThrRS:ATP structure.

tRNA-induced movements

The binding of tRNA triggers a displacement ofthe four mobile loops. The movement of the threo-nine loop is similar to that induced by the aminoacid, albeit of smaller amplitude. The hydroxylgroup of Tyr462, which hydrogen-bonds the a-amino group in the threonine complex, moves by2 A with respect to the apo structure and interactshere with the 20 hydroxyl of the terminal riboseA76 (Figure 4(c)). Similarly, Arg375 of the motif 2loop, which in the ATP complex is in contact withthe g-phosphate, now switches to C74 recognitionthrough two base-specific hydrogen bonds. In theATP loop, Arg476 binds the phosphate group ofG71, instead of associating with Glu527 in the sys-tem-conserved salt bridge seen in the apo andthreonine complexes. Finally, the ordering loop isalso involved in direct interactions with the tRNA(Figure 4(c)). Tyr313 stacks on the terminal adeno-sine A76. The aromatic character of this residue,which allows p interactions with the base, is con-served in the GlyRS, SerRS and ProRS systems.Further stabilization of A76 is provided by His309,interacting with the ribose O20 and by the main-chain carbonyl of residue 316, interacting withthe amine group of the adenine base. These

Figure 5. Annealed omit maps in the active site of ThrRS from S. aureus, contoured at 1.5s. The substrates and thesolvent molecules were omitted from the simulated annealing refinement and the calculation of the electron densitymaps. (a) SaThrRS:ATP structure, subunit A of the dimer, showing ATP in the bent conformation. (b) SaThrRS:ATPstructure, subunit B. (c) and (d) SaThrRS þ ThrAMS structure in subunits A and B of the dimer, respectively. For con-sistency, the amino acid numbering of E. coli ThrRS is used here for S. aureus (the positions shown are occupied by thesame amino acid in the two systems, except for Glu484, replaced by a triptophan in E. coli).


observations, made for the E. coli species, can beextended to S. aureus, since all the residuesinvolved are conserved (with the exception ofArg476, replaced by Lys482 in S. aureus).

Discussion

The structure of SaThrRS with ATP presents inboth subunits an electronic density that is stronglysuggestive of a threonine molecule. If the two sub-strates, ATP and threonine, are present in the activesite, with the necessary Mg2þ, one would expect thefirst step of the aminoacylation reaction, the for-mation of threonine adenylate, to take place. How-ever, the reaction here is blocked. A possibleexplanation is that, in the crystal, packing effects pre-vent a productive reaction. Similar situations wereencountered in LysRS, with lysine and unreactedATP simultaneously present in the active site16 orE. coli AspRS, where aspartyl adenylate was foundin the presence of tRNA, showing the situationbefore the second step of the aminoacylationreaction.12 The presence of threonine should not beinterpreted as indicative of a high affinity of ThrRSfor threonine. With a Km value of 0.11 mM forthreonine,9 ThrRS has no better affinity for its sub-strate than other synthetases. Rather, it suggests theselection of the threonine-bound form in the crystal-lization process. Addition of threonine (or thrAMS)was indeed required for obtaining E. coli DN-ThrRScrystals in the absence of tRNA. In the case of S. aur-eus ThrRS, no threonine was added, but some threo-nine was most probably carried over during thepurification process.

The catalytic domain of ThrRS shows concertedmotion of different regions of the active site.Amino acid binding promotes the movement ofthree regions among which is the threonine loop,directly involved in amino acid recognitionthrough Tyr462. The displacement of this loopbrings the catalytic Lys465 at binding distance ofthe a-phosphate of ATP. The binding of this sub-strate produces the large movement of the ATPloop. ATP also interacts with the motif 2 loop.Through Arg363, this loop senses the presence ofboth ATP and threonine, and thus plays an essen-tial role in the first step of the aminoacylation reac-tion. This analysis suggests synergy rather thanneed for an ordered binding of the small sub-strates, since the positioning of either amino acidor ATP facilitates the binding of the other by con-tributing to its optimal-binding site.

The tRNA binds to all of the four mobile regionsand takes advantage of the conformational changescaused by the other two substrates. The correctpositioning of the acceptor arm is assisted by aseries of conformational switches, each one in adifferent loop. Tyr462 of the threonine loopswitches from the recognition of the amino acid tothat of the 20 hydroxyl of the terminal adenosine.This tyrosine is therefore a key actor of the reac-tion. It stabilizes the ribose of the terminal adeno-

sine and helps to fix its position for the secondstep of the reaction. Not surprisingly, it is con-served in the class IIa GlyRS, HisRS and ThrRS sys-tems. In other class II synthetases, this isperformed by residues from the TXE loop, like theconserved threonine in SerRS interacting with thea-amino group of the seryl adenylate analogue,23

or the equivalent serine in AspRS, which bindsthe ribose of A76.12 Another switch is found inmotif 2, with Arg375 changing from the g-phos-phate to the tRNA-binding position. This residuethus senses the departure of the pyrophosphateafter completion of the amino acid activation. Athird switch relies on Arg476, in the ATP loop.This residue, released from the conserved saltbridge upon ATP binding, is free to bind the phos-phate chain of the tRNA at the level of C74. Theelimination of pyrophosphate is necessary to makeroom for the 30 end of the tRNA and to leaveArg375 free to bind C74. This is consistent withthe observation that AMP was necessary to obtaincrystals of the active ThrRS:tRNA complex(A.C.D.B., unpublished observation; actually, ATPwas added, but the presence of AMP in the activesite10 indicated that hydrolysis had occurred, prob-ably due to a contaminant of the ThrRS prep-aration, as shown later in the case of theThrRS:operator complex26). In class II synthetases,the binding of the tRNA is not necessary for thefirst step of the reaction, in contrast to some class Ienzymes.24 Conversely, it appears from the presentanalysis that the conformational changes due tothe binding of the small substrates are required toallow a productive binding of the tRNA 30 end inthe active site.

A conformational switch was described in SerRSfor the motif 2 loop, which displays different confor-mations depending on the presence of ATP ortRNA.18 In ThrRS, three out of the four mobileregions of the catalytic domain contain key residues,which switch conformation throughout the catalysisand play an essential role in the dynamics of the reac-tion. The fourth mobile region, the ordering loop,contributes to closing the active site cleft once thethree substrates are in place. This capacity of closingand opening the active site may play a role in therelease of the reaction products.

Class II synthetases are obligate dimers. Thedimeric state, characterized by a largely conservedinterface containing the consensus motif 1, may bethe scaffold of a functional interdependence of thetwo subunits, as has been shown in the case ofAspRS.25 The conformational changes describedhere do not provide further evidence of directinter-subunit interactions. However, and in con-trast to other synthetases like AspRS, where eachmonomer interacts with only one tRNA molecule,each tRNAThr binds both monomers of ThrRS,10,26

and it is tempting to look for some tRNA-inducedcooperative effect. The phosphate and O30 of U66make cross-subunit contacts with Asn324 andArg325, respectively (Figure 4(c)). These residuesare part of a region known as small interface (SI)


motif, which flanks the ordering loop and has beenproposed to play a role in the dimer communi-cation in HisRS and ProRS.7,8 The adjacent residueGlu323 is involved in a salt bridge with the motif2 loop Arg377 of the other monomer, in all ThrRSstructures but in the complex with tRNA. Thebinding of tRNA by one monomer, by breakingthis salt bridge, may thus modify the position andflexibility of the ordering loop of the other mono-mer and consequently affect the binding or releaseof the substrates in the second active site. Cross-subunit interactions with the tRNA have also beenidentified in ProRS and SerRS. In the case ofHisRS, the structure of the complex with tRNA isnot known, but there is a system-conserved cross-subunit salt bridge made by the homologous resi-dues (Asp70 and Arg122 in T. thermophilus HisRS).This type of tRNA-induced inter-subunit com-munication could thus constitute another func-tional link between members of subclass IIa.

Aminoacylation has to reconcile complexity (atwo-step process dealing with three substrates)and efficiency. To perform this task efficiently,class II synthetases have adopted both the lock-and-key and the induced-fit mode of recognition.Their ligand-binding pockets satisfy a balancebetween flexibility and rigidity in order not onlyto recognize and bind the substrates, but also torelease the reaction product, the aminoacylatedtRNA, at the lowest possible energetic cost. Afterthe initial discrimination performed rigidly via thezinc ion, we see here that ThrRS makes mainlyuse of induced-fit mechanisms during the catalyticprocess, as also do the other class IIa synthetases,HisRS7,8 and ProRS.8 Building the active site upona small number of highly selected and mobile resi-dues provides efficiency because each of them inthe way of a switch probes two substrates andallows the sensing of the status of the reaction.There is an intrinsic thermodynamic penaltyattached to induced fit, but this is compensated bythe fact that wasting of substrates is avoided, asseen in the classical hexokinase case.27 While theside-chain of the amino acid is recognized in thelock-and-key mode, all the mobile parts of theactive site are involved in the recognition of invari-able parts of the substrates (ATP, the end of theacceptor arm of the tRNA, and the carboxylateand amino moieties of the amino acid). Notably,Tyr462, which is responsible for the recognition ofthe amino group, undergoes the same displace-ment in the presence of the non-cognate serinesubstrate.9 Similarly, in M. thermautotrophicusProRS, a number of loops move or become orderedin the same way upon binding of prolyl-, cysteinyl-or alanyl-adenylate, which can all be accomodatedin the active site pocket.28 These observationssuggest that induced fit is not a determinant fordiscrimination, which is rather based on rigid rec-ognition of the amino acid side-chain. A good illus-tration is provided by the class IIb AspRS, wherean extensive hydrogen-bond network blocks theconformation of a few key residues involved in

the recognition of the aspartic acid side-chain.29 Indifficult cases, like the discrimination of threoninefrom serine by ThrRS, the direct recognition iscomplemented by an independent editing reaction.

Materials and Methods

Expression and purification

SaThrRS (Mr 74,460) overexpressed in E. coli was pro-vided by SmithKline Beecham Pharmaceuticals as a crudeextract after culture, extraction and a first step of chroma-tography on DEAE-Sepharose. The purification was com-pleted by an additional anion-exchange chromatographyon Q-Sepharose and a hydrophobic chromatography stepon phenyl-Sepharose in the presence of ammonium sul-fate. Biological activity was assessed, like that of the E. colienzyme, by a standard aminoacylation assay in 50 mMHepes–NaOH (pH 7.7), 20 mM MgCl2, 5 mM b-mercap-toethanol, 30 mM KCl, 10 mM ATP with 50 mM [14C]threo-nine. Pure E. coli tRNAThr (4 mM) was used for this assay,and the enzyme concentration was 200–300 nM.

Crystallization

Crystallization experiments were conducted using thehanging-drop technique at 4 8C. The drop solution wasa 1:1:1 mixture of the protein at 15 mg/ml (in buffer20 mM K Hepes (pH 7.2), 2 mM MgCl2, 100 mM KCl), aligand-containing solution (10 mM MgCl2 and 10 mMATP or 10 mM MgCl2 and 1 mM ThrAMS), and the wellsolution (12–14% (w/v) of PEG 8000, 50 mM of Tris·HClbuffer at pH 7.5–8.5 and ammonium acetate or potass-ium chloride in the 0.2–0.4 M range). Needle-shapedcrystals appeared after two to three days, the largest hav-ing approximate dimensions of 5 mm £ 30 mm £ 300 mm.

Data collection and processing

Crystals were placed for two minutes in the wellliquor solution supplemented with glycerol in increasingconcentrations of 5, 10, 15 and 22% (v/v) and then flashcooled in liquid ethane. The crystals of the two com-plexes are isomorphous and belong to the orthorhombicspace group P212121 (Table 3). They contain one dimerper asymmetric unit, and the estimated solvent contentis 70%. Diffraction data were collected at the ESRF, Gre-noble, on the beamlines BM30 for the ThrRS-ATP com-plex and ID14/EH3 for the ThrRS-AMS complex, bothequipped with a MAR CCD detector. All data were pro-cessed with the programs DENZO and SCALEPACK.30

Structure solution and refinement

The structure of SaThrRS:ATP was solved by molecu-lar replacement using the AMoRe program suite31 andthe coordinates of the EcThrRS-tRNA structure10 as amodel. The dimeric catalytic core and each of the N-terminal domains of the dimer were fitted separately asrigid bodies, resulting in an R-value of 51.1% in the 10–4.5 A resolution range. A model of SaThrRS was thenbuilt with the program O32 and refined with CNS, usingthe torsion-angle molecular dynamics option.33 Arandom sample of 5% of the reflections was used tomonitor the course of the refinement. The two monomersof the dimer present in the asymmetric unit showed


significant differences and were therefore built andrefined independently. A similar strategy of model build-ing and refinement process was used for the SaThrRS:ThrAMS structure. The results of the refinements aresummarized in Table 3.

Several regions, especially in the N-terminal part ofthe molecule, are poorly defined in the two crystal struc-tures. This is probably due to the inherent flexibility ofthe enzyme and the lack of stabilization by the tRNA. Itis consistent with the difficulty of crystallizing the com-plete enzyme not complexed by tRNA: we never suc-ceeded in obtaining crystals of acceptable diffractingquality of the whole E. coli enzyme in the apo state orwith the small substrates only. Most regions involved incatalysis are however quite well ordered in the crystalsand show a clear electron density.

Atomic coordinates

The coordinates of the S: aureus ThrRS structures havebeen deposited in the RCSB Protein Data Bank. Theaccession code is 1NYR For ThrRS:ATP:threonine and1NYQ for ThrRS:ThrAMS.

Acknowledgements

This work was supported by EU (project No.BIO4-97-2188). A.T.-L. is a recipient of a scholar-ship from Consejo Nacional de Ciencia y Tecnolo-gia (Mexico). We are very grateful to DrP. O’Hanlen from SmithKline Ltd, who provided

ThrRS from Staphylococcus aureus. We also thankthe staff of the ESRF at Grenoble for their helpduring the data collections.

References

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Table 3. Data collection and refinement statistics

SaThrRS:ATP SaThrRS:AMS

Space group P212121 P212121

a (A) 104.13 104.28b (A) 122.52 122.73c (A) 148.66 149.94

Data statisticsa

Wavelength (A) 0.9797 0.9310Resolution limits (A) 25–2.8 (2.87–2.8) 20–3.2 (3.31–3.2)Observed reflections 160,435 125,027Unique reflections 46,973 (2998) 32,055 (3183)Redundancy 3.4 3.9Rmerge (%) 3.6 (29.0) 5.1 (28.9)kI/s(I)l 19.1 (3.8) 18.6 (4.4)Completeness (%) 98.3 (95.7) 98.2 (99.6)

Refinement statisticsResolution limits 15–2.8 15 –3.2Reflections in working set 44,307 27,572Reflections in test set 2311 3076R-factor (%) 23.9 20.5R-free (%) 31.3 28.5rmsd of bond lengths (A) 0.012 0.012rmsd of bond angles (deg.) 1.7 1.7Mean protein B-factor (A2) 73.9 70.6

Ramachandran plotResidues in most favored regions (%) 75.2 74.2In additionally allowed regions 21.3 22.7In generously allowed regions 3.2 2.8In disallowed regions 0.3 0.3

a Values in brackets are for the highest resolution range.


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Edited by R. Huber

(Received 27 February 2003; received in revised form 3 June 2003; accepted 4 June 2003)


conformational movements and cooperativity upon amino acid, atp and trna binding in threonyl-trna...

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