The Structure of Yeast Glutaminyl-tRNA Synthetase and ... · The Structure of Yeast Glutaminyl-tRNA Synthetase and Modeling of Its Interaction with tRNA Thomas D. Grant 1, Joseph

The Structure of Yeast Glutaminyl-tRNA Synthetase andModeling of Its Interaction with tRNA

Thomas D. Grant1, Joseph R. Luft1,2, Jennifer R. Wolfley1, Mary E. Snell1, Hiro Tsuruta3, Stephanie Corretore4, Erin Quartley4,Eric M. Phizicky4,5, Elizabeth J. Grayhack4,5 and Edward H. Snell1,2

1 - Hauptman Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203, USA2 - Department of Structural Biology, State University of New York at Buffalo, 700 Ellicott Street, Buffalo, NY 14203, USA3 - Stanford Synchrotron Radiation Lightsource, 2575 Sand Hill Road, Menlo Park, CA 94205, USA4 - Department of Pediatrics, University of Rochester Medical Center, Rochester, NY 14642, USA5 - Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA

Correspondence to Edward H. Snell: Hauptman Woodward Medical Research Institute, 700 Ellicott Street, Buffalo,NY 14203, USA. [email protected]://dx.doi.org/10.1016/j.jmb.2013.03.043Edited by J. Doudna

Abstract

Eukaryotic glutaminyl-tRNA synthetase (GlnRS) contains an appended N-terminal domain (NTD) whoseprecise function is unknown. Although GlnRS structures from two prokaryotic species are known, noeukaryotic GlnRS structure has been reported. Here we present the first crystallographic structure of yeastGlnRS, finding that the structure of the C-terminal domain is highly similar to Escherichia coli GlnRS but that214 residues, including the NTD, are crystallographically disordered. We present a model of the full-lengthenzyme in solution, using the structures of the C-terminal domain, and the isolated NTD, with small-angleX-ray scattering data of the full-length molecule. We proceed to model the enzyme bound to tRNA, using thecrystallographic structures of GatCAB and GlnRS–tRNA complex from bacteria. We contrast the tRNA-boundmodel with the tRNA-free solution state and perform molecular dynamics on the full-length GlnRS–tRNAcomplex, which suggests that tRNA binding involves the motion of a conserved hinge in the NTD.

© 2013 Elsevier Ltd. All rights reserved.

Introduction

Aminoacyl-tRNA synthetases are required in allthree domains of life for covalently attaching aminoacids to their cognate tRNA molecules for use inprotein synthesis.1 These enzymes are separatedinto two structural classes,2,3 class I and class II,which operate via a two-step reaction. First, afterbinding ATP and the amino acid, an aminoacyl-adenylate is formed, followed by esterification of theamino acid moiety to either the 2′ OH (class I) or 3′OH (class II) of tRNA. In addition, eukaryotic tRNAsynthetases often contain additional domainsappended to their N-terminal or C-terminal endsthat are not found in their prokaryotic homologs.4

These domains are known to have various roles,including nucleic acid binding, protein–protein in-teractions, and hydrolytic editing mechanisms1;

0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserve

however, many of these domains are of unknownfunction. While structures of several eukaryotic tRNAsynthetases have been determined,5 virtually all ofthese lack the appended domain. The structures ofsome appended domains have been determined inisolation, but few models of full-length eukaryotictRNA synthetases exist.While in most cases one synthetase exists for each

amino acid, an exception occurs for glutamine andasparagine.6 In eukaryotes and some bacteria, thetraditional pathway of aminoacylation exists forglutamine, in which glutaminyl-tRNA synthetase(GlnRS) binds to tRNAgln, glutamine, and ATP andfirst forms a glutaminyl-adenylate molecule that isthen covalently attached to the 3′-end of tRNAgln withthe release of AMP. A different pathway exists inmost bacteria and all archaea, where a non-discriminating glutamyl-tRNA synthetase (GluRS)

d. J. Mol. Biol. (2013) 425, 2480–2493

http://dx.doi.org/10.1016/j.jmb.2013.03.043


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2481The Structure of Yeast Glutaminyl-tRNA Synthetase

attaches glutamic acid to both tRNAglu and tRNAgln.The misacylated Glu-tRNAgln is then converted toGln-tRNAgln by the GatCAB amidotransferase en-zyme in bacteria and some archaea or by the GatDEamidotransferases in other archaea. GatCAB per-forms a similar function for asparaginyl-tRNAasn.Since this indirect pathway for aminoacylation existsin most prokaryotes, GlnRS is primarily a eukaryoticenzyme, and its presence in a small number ofbacteria is believed to have occurred through ahorizontal gene transfer event.7 Nonetheless, thereare significant differences between the prokaryoticand eukaryotic GlnRS enzymes, as described below.Structural data for two prokaryotic GlnRS species

exist,8,9 yet no structure has been reported for anyfull-length eukaryotic GlnRS. Saccharomyces cere-visiae GlnRS (Gln4) is an 809-residue protein thatcontains a 215-residue domain appended to its N-terminus. This domain is nearly ubiquitous amongeukaryotic GlnRS species but absent in prokaryotichomologs.10 Recently we described the structure ofthe N-terminal domain (NTD) of Gln4, revealing that ithas an extraordinary structural resemblance to theregion of the B subunit of the GatCAB amidotransfer-ase (GatB)11 that binds to tRNAgln.12 Although

deletion of the NTD distinctly affects catalytic activity,growth in yeast, and tRNA binding,12 the manner inwhich tRNA binding occurs is still unknown.Here we present the first crystallographic structure

of the C-terminal domain (CTD) of Gln4 from crystalsof the full-length protein. Based on this structure, thestructure of the isolated NTD,12 and small-angleX-ray scattering (SAXS) data of the full-lengthenzyme, we present a model of the full-lengthenzyme in solution. We extend this to model thefull-length enzyme bound to tRNAgln from thecrystallographic structures and homology withknown transamidosome and GlnRS–tRNA complexstructures11,13 yielding new insights into the struc-tural rearrangements occurring in eukaryoticGlnRS–tRNAgln complex formation.

Results and Discussion

Overall structure

We solved the crystal structure of Gln4 to 2.15 Åresolution [Protein Data Bank (PDB) ID: 4H3S] by

Fig. 1. X-ray crystal structure ofGln4. Cartoon representation of theGln4 structure is shown color-codedaccording to domains and inser-tions relative to E. coli GlnRS.Residues 1–214 are missing in theelectron density. Domains are la-beled with corresponding aminoacid numbers.

Table 1. Crystallographic data collection parameters andstatistics

Beamline SSRL BL 11-1

Wavelength (Å) 1.169Space group P3121Unit cell parametersa, b, c (Å) 176.61, 176.61, 72.19α, β, γ (°) 90, 90, 120Resolution limits (outer shell) (Å) 52.49–2.15 (2.227–2.15)No. of unique reflections 70,276 (6963)Completeness (%) 99.86 (99.84)Rsym (%) 0.068 (0.348)⟨I/σ(I)⟩ 23.26 (2.98)ReflectionsWorking 70,276 (6963)Test (%) 5Atoms 10,566Protein 5031Water 447Ligands 75Rfactor 0.160 (0.222)Rfree 0.175 (0.242)RMSDBonds (Å) 0.003Angles (°) 0.73Average B-factorOverall 48.10Macromolecules 47.40Solvent (water) 54.20Ramachandran analysis (%)Most favored regions 99Outliers 0.17Generously allowed regions 0Disallowed regions 0Clash score 5.27PDB code 4H3S

2482 The Structure of Yeast Glutaminyl-tRNA Synthetase

single-wavelength anomalous dispersion with a zincanomalous signal identified from an initial excitationscan, coupled with molecular replacement using thecrystal structure of the Escherichia coli GlnRS as atemplate (Fig. 1 and Table 1). Of the 809 amino acidsin the primary sequence of Gln4, ~30% were notresolved in the electron density. The unresolvedregions are residues 1–187 (which correspond to theNTD), residues 188–214 (which encode a noncon-served 26-residue region that is predicted to beunstructured and links the NTD and CTD; Fig. S1),and residues 672–678 (which encode a disorderedloop within the CTD). The presence of the NTD inpurified protein and in the crystals was confirmed byexpressing Gln4 with a His6 tag on its N-terminus,followed by purification using Ni2+ affinity chroma-tography, crystallization, and antibody detection withanti-His antibody on dissolved crystals. Residues 1–214 were still unresolved in structural data fromthese crystals.Since the NTD of Gln4 is known to be structured12

and is present in the full-length protein in the crystal,but no electron density is observed for this region,we conclude that the NTD is crystallographicallydisordered (i.e., the position and/or orientation of theNTD varies between unit cells). It seems likely thatthe NTD is located in large solvent channelsapproximately 145–175 Å wide observed along thez-axis (Fig. S2). Consistent with this, we note that thechannels can comfortably accommodate the struc-ture of the NTD,12 which has dimensions of~25 Å × 25 Å × 75 Å and a macromolecular vol-ume of ~25,000 Å3. Moreover, the first residueresolved in the electron density, Arg215, is locatedadjacent to the solvent channels, and the precedingresidues, 188–214, are predicted to be unstructured(Fig. S1). It is therefore likely that this region is thecause of the crystallographic disorder of the NTD.Based on the considerations presented above,

using the naming convention previously describedfor E. coli GlnRS,14 the overall structure of Gln4consists of a folded NTD (residues 1–187); apositively charged 26-residue region linking theNTD and CTD (residues 188–214); a catalyticdomain consisting of a Rossmann fold domain(residues 215–324 and 439–498), an acceptorstem binding domain (residues 325–438), and ahelical subdomain (residues 499–567); and ananticodon binding domain consisting of proximal(residues 568–574 and 690–809) and distal (resi-dues 575–689) β-barrels (Fig. 1). The structure ofthe CTD of Gln4 (residues 215–809) is highly similarto E. coli GlnRS (PDB ID: 1NYL; RMSD = 1.86 Å15)but has multiple insertions relative to the bacterialhomolog. The Gln4 catalytic domain (residues 215–567) is highly conserved in sequence and structure,showing an E value of 1 × 10−101 relative to the E.coli GlnRS catalytic domain using PSI-BLAST andan RMSD of only 1.03 Å. The Gln4 anticodon

binding domain (residues 568–809) shares moder-ate sequence homology but high structural conser-vation showing an E value of 5 × 10−32 and anRMSD of 1.13 Å. The structures of the Gln4 CTDand a second bacterial homolog, Deinococcusradiodurans GlnRS (PDB ID: 2HZ7),8 are alsosimilar with an RMSD of 1.62 Å.A detailed comparison of the Gln4 CTD structure

with the unliganded (PDB ID: 1NYL)15 and tRNA-bound (PDB ID: 1QTQ)13 E. coli GlnRS structuresrevealed both similarities and differences in theindividual residues previously found to be importantsubstrate recognition elements in E. coli GlnRS.16,17

The ATP-binding pocket formed by the HIGH andMSK (LSK in Gln4) is similar to that found in E. coliGlnRS, which are signature sequences conservedacross class I aminoacyl-tRNA synthetases.1 Thereplacement of Met268 in E. coli GlnRS with Leu496in Gln4 does not appear to significantly change thestructure of this pocket. In E. coli GlnRS, residuesArg133, Arg192, Lys194, Thr68, and Asn69 wereshown to be important in forming the hairpin loop ofthe tRNAgln CCA tail that is necessary to bring the 2′hydroxyl of A76 into proximity with the glutamineadenylate molecule for glutamine transfer to tRNA.16


In Gln4, these residues (Arg357, Arg421, Leu422,Thr293, and Asn294, respectively) are conserved inposition and orientation and therefore are likely tocause the CCA tail to form a similar hairpin loop. Inthe anticodon binding region of E. coli GlnRS,Arg341 interacts with anticodon base U35, resultingin distant, interdomain communication to the activesite necessary for glutamine recognition.15 Thecorresponding residue in Gln4, Arg568, is in thesame orientation and therefore could lead to similarinteractions with U35. In E. coli GlnRS, Lys401 andArg402 interact with G36, which is the discriminatingbase of the anticodon, and are crucial for recognitionof cognate tRNAgln and discrimination againstnoncognate tRNAglu. In Gln4, while Arg402 isconserved as Arg631, Lys401 is replaced byPhe630. While both lysine and phenylalanine arelarge residues, it is unknown if the loss of thepositively charged side chain results in differentinteractions with G36 in Gln4.Multiple sequence analysis among eukaryotic

GlnRS species revealed three insertions relative toE. coli GlnRS (Figs. 1 and 2). Insertion 1, located atresidues 234–250 of Gln4, is present in all eukary-otes, including those species lacking an NTD,although its length and sequence are not con-served among different kingdoms. Comparison withthe E. coli GlnRS structure reveals that thisinsertion is part of the Rossmann fold domain inthe CTD. Insertion 2 is located at residues 364–368and is situated in a zinc binding motif consisting ofCys346-X-Cys348-X24-Cys372-X-His374 (Fig. S3).The zinc binding motif and Insertion 2, which arealways found together, are present in fungal GlnRS

Fig. 2. Domain architecture of GlnRS. Domains for GlnRS athe bacterium D. radiodurans, and archaeal GluRS. Major domdomain, and the unique CTD appended to the D. radioduranslabeled along with the zinc coordinating residues of the zincarchaeal GluRS.

species but are absent in other eukaryotes.Insertion 3 is a loop located at residues 751–770and is also only conserved among fungi and absentin other eukaryotes.GlnRS selects both the correct amino acid

substrate, glutamine, and the cognate tRNAgln

molecule. To gain insight into the tRNAgln discrim-ination mechanism in Gln4, we performed furthersequence analysis incorporating the archaeal non-discriminating GluRS, an evolutionary precursor ofGlnRS that has the ability to recognize both tRNAgln

and tRNAglu.18 This revealed that Insertion 2 is partof a larger nine-residue loop, Loop 1, which is absentin archaeal GluRS (Fig. 2). In E. coli GlnRS, Loop 1is only four residues long and has been suggested toprovide the ability to discriminate against the G1:C72base pair in tRNAglu and instead only recognizestRNAgln by disrupting the weak U1:A72 base pair.18

Our results suggest that Loop 1 may play a differentrole in fungi and other eukaryotes than it does inbacteria. Although Loop 1 is present in Gln4, it differsin amino acid sequence compared to E. coli GlnRSand, with Insertion 2, is five residues longer.Additionally, while the first base pair of tRNAgln isU1:A72 in E. coli, the first base pair of tRNAgln is G1:C72 in fungi and other eukaryotes. It is thereforeunlikely that the extended Loop 1 seen in Gln4functions similarly to E. coli GlnRS Loop 1 bydisrupting the first base pair, since if the extendedLoop 1 in Gln4 were to disrupt the strong G1:C72base pair in tRNAgln it might also disrupt the weakU1:A72 base pair in tRNAglu and fail to discriminatebetween them. While our observations are based onlength, it is unknown whether sequence-specific

re shown for fungi, eukaryotes (other than fungi), bacteria,ains are labeled NTD, catalytic domain, anticodon bindingGlnRS. Insertion 1, Insertion 2, Insertion 3, and Loop 1 arebinding motif, which is present only in fungal GlnRS and

image of Fig.�2


contacts in Loop 1 contribute tRNA discriminationability to Gln4. However, in all eukaryotes other thanfungi, Loop 1 is entirely absent (Fig. 2) and thisregion of eukaryotic GlnRS more closely resemblesarchaeal GluRS, suggesting that Loop 1 is not likelyto be a critical tRNA discriminatory motif in eukary-otes with the possible exception of fungi.

Motion of NTD in solution is limited in theabsence of tRNA

We collected SAXS data on full-length Gln4(Fig. S4) because SAXS is a solution-basedtechnique that is sensitive to the overall shape andsize of a molecule and can also provide insight intoprotein dynamics.19,20 Analysis of the pair distribu-tion function (the distribution of interatomic dis-tances) yields a maximum particle dimension of~147 Å and a radius of gyration (Rg) of 43.51 Å, inclose agreement with that estimated by Guinieranalysis,21 43.32 Å. This is significantly larger thanthe maximum dimension and Rg of the CTD from thecrystal structure (108 Å and 32.3 Å, respectively).The increased values are consistent with thepresence of an appended NTD. The SAXS data offull-length Gln4 suggest an elongated particle shapedue to the large maximum dimension compared tothe Rg and the left-skewed pair distribution function(Fig. S4d).Since the crystallographic data showed that the

NTD was disordered, we used the SAXS data toprobe the motion of the NTD in solution. Informationabout protein flexibility can be obtained from aPorod–Debye analysis of SAXS data, where thedecay in intensity is studied as a function of angleand reported as the Porod exponent.22 In cases ofglobular, rigid molecules, the Porod exponent willapproach 4, whereas for flexible, random chains, itwill approach 2. A Porod–Debye analysis of full-length Gln4 resulted in a Porod exponent of 3.4,suggesting a well-folded protein with mild flexibility,consistent with the crystallographic data. The totalvolume occupied by the protein can be measuredfrom the SAXS data and is known as the Porodvolume.23 The Porod volume of full-length Gln4 is~153,000 Å3, corresponding to an estimated mo-lecular mass of 92.6 kDa,22 similar to the molecularmass estimated from the forward scattering [I(0)] of89.1 kDa and the expected molecular mass of93.1 kDa (these data also confirm that the full-lengthprotein is intact). This demonstrates that the volumeoccupied by the NTD is approximately limited to thesize of the domain and does not occupy a largerregion of conformational space.Ten ab initio envelope reconstructions of full-

length Gln4 were created and averaged, exhibitingan average normalized spatial discrepancy (NSD) of0.633 ± 0.022. NSD is a measure of the varianceamong a set of multiple SAXS envelopes. NSD

values less than 1 reflect uniqueness and lowflexibility,24 whereas values greater than 1 reflectan inability to describe the protein using one uniqueshape. To generate a rigid-body model, we created10,000 random conformations of full-length Gln4 bytreating the NTD and CTD crystal structures asindependent rigid bodies and allowing the linker(residues 188–214) to be flexible. The model whosecalculated scattering best fit the experimental SAXSdata was selected from this pool. This model isshown in Fig. 3 superposed onto the ab initioenvelope. The rigid-body model fits well to theexperimental scattering data with χ2 = 1.82. Thelocation of the NTD of Gln4 appears to be similar tothe appended domain modeled in the hybridstructure of GlnRS from D. radiodurans.8 D. radio-durans is unique because it is the only bacteriaknown to contain a GlnRS with an appendeddomain. However, the appended domain of D.radiodurans GlnRS is located on the C-terminus,downstream of the core enzyme (Fig. 2), andcontains no sequence homology to the appendeddomain of eukaryotic GlnRS, which is located on theN-terminus, upstream of the core enzyme. Thesimilar spatial location of both appended domains,despite being extensions at different termini of thecore enzyme, is made possible by the much longer80-residue linker in D. radiodurans GlnRS, com-pared to the 26-residue linker in Gln4. Additionally,the appended domain of D. radiodurans GlnRS wasshown to cross-react with an antibody that recog-nizes the GatB subunit of the GatCAB amidotrans-ferase used in the indirect pathway of Gln-tRNAgln

formation used in most prokaryotes.8 Our previousstudy reported a high degree of structural similaritybetween the Gln4 NTD and the GatB subdomain,12

suggesting that the appended domains of GlnRSfrom eukaryotes and D. radiodurans perform similarfunctions and are likely an instance of convergentevolution.Ab initio reconstructions and rigid-body modeling

do not take into account the dynamic informationpresent in SAXS data but represent an average of allconformations present in solution. Ensemble model-ing can overcome this by representing a proteinstructure as an ensemble of multiple conformations.Using the ensemble optimization method24 withmultiple conformations of the NTD did improve theoverall fit to the scattering data (χ2 = 1.75); however,the improvement was only marginal. The Rg distri-bution of conformers present in the ensemble wassimilar to that seen for simulated data of the rigid-body model alone, suggesting little flexibility. Thelimited flexibility from the Porod–Debye analysis, thelow NSD for multiple ab initio envelope reconstruc-tions, and the marginal improvement from anensemble model compared to the rigid-body modeldemonstrate that the mobility of the NTD in solutionis limited.

Fig. 3. SAXS rigid-body model offull-length Gln4. (a) Two orienta-tions rotated 90° relative to eachother are shown in cartoon repre-sentation and colored according tosecondary structure and super-posed onto the ab initio envelopeshown in gray. (b) The calculatedscattering of the rigid-body model(black continuous line) fit to theexperimental SAXS data (graycircles).


A model of full-length Gln4 bound to tRNAgln

suggests a substantial conformational changeof the NTD

We used the structure of E. coli GlnRS bound totRNA13 and the structure of Thermotoga maritimaGatCAB bound to tRNA11 to obtain a model of full-length Gln4 bound to tRNA. First, the structure ofE. coli GlnRS bound to tRNAgln (PDB ID: 1QTQ)was superposed onto the Gln4 CTD (residues 215–809). The structure of the tRNA molecule was thenextracted, providing an initial model of tRNAgln

complexed with the Gln4 CTD. Since the nucleotide

sequence of E. coli tRNAgln differs from yeasttRNAgln, the tRNA molecule was computationallymutated to match that of yeast tRNAgln and ageometric minimization was performed usingModeRNA.25 Our model contains similar contactsbetween the tRNAgln anticodon and the Gln4anticodon binding domain that were shown to beimportant identity elements in E. coli GlnRS9,13 dueto the high degree of structural similarity betweenE. coli GlnRS and the Gln4 CTD.To correctly place the NTD in the model, we

utilized known structural homology of the NTD withsubunit B of the GatCAB amidotransferase (GatB)

image of Fig.�3


from T. maritima (PDB ID: 3AL0), which was solvedin complex with tRNAgln. The tail subdomain(residues 119–187) of the Gln4 NTD was super-posed to the tail subdomain of GatB due to the highlevel of structural homology between these tworegions.12 Finally, MODELLER26 was used for denovo modeling of the predicted flexible linker(residues 188–214) (Fig. 4).Our model of full-length Gln4 bound to tRNAgln

shows a significant change in the NTD positionwhen compared to the tRNA-free, SAXS-derivedconformation (Fig. 5a). The model shows an ~160°rotation and an ~40-Å translation of the NTD withrespect to the solution conformation according to ananalysis by DynDom.27 To determine if this newconformation of the NTD exists in solution, wesimulated the SAXS profile from our model of Gln4bound to tRNA, after removing the tRNA molecule,and compared it to the experimental SAXS data.Fitting the simulated scattering of only the proteinportion of the model of the protein–tRNA complexto the experimental SAXS data resulted in a poorfit, yielding χ2 = 12.25 compared to 1.82 for therigid-body model created using the experimentalSAXS data (Fig. 5b). The limited flexibility of theNTD, coupled with the poor fit of the simulatedscattering of the protein portion of the model boundto tRNAgln, suggests that, without tRNA bound, thisconformation does not exist in solution.

Fig. 4. Homology model of full-length Gln4 bound to tRNAgln

and rotated model showing gap between NTD helical subdom

Conformational change in NTD subdomains ispredicted upon interaction with tRNAgln

In the compiled model of Gln4 bound to tRNA, theNTD subdomains appear to be in an “open”conformation resulting in the helical subdomain(residues 1–110) being too distant from the tRNAmolecule to form stable contacts (Fig. 4b). Thisconformation more closely resembles that observedin the Staphylococcus aureus GatCAB structuresolved without tRNA bound12 than the conformationseen in tRNA-bound T. maritima GatCAB. Thus, weconsidered that the molecule might undergo aconformational change upon tRNA binding. Toprobe the dynamics of full-length Gln4 in complexwith tRNAgln, we carried out 70 ns of moleculardynamics simulation. The model stabilized withinapproximately 10 ns of simulation time andremained relatively unchanged for the remaining60 ns (Fig. S5), showing that the simulation timecaptured the relevant dynamics.The resulting molecular dynamics trajectory

(Figs. 6 and 7 and Fig. S5) shows that the helicalsubdomain of the NTD rotates about a conservedhinge by 22.9°, forming electrostatic interactionsbetween the positively charged side chains of lysineresidues 19, 20, and 26, which have been previouslyimplicated in tRNA binding integrity,28 and thenegatively charged phosphate backbone of tRNA.

. (a) Full-length Gln4 shown bound to tRNAgln. (b) Enlargedain and tRNA molecule.

image of Fig.�4

Fig. 5. Comparison of NTD position before and after tRNA binding. (a) Gln4 is shown in cartoon representation. TheNTD position prior to tRNA binding determined by SAXS rigid-body modeling is shown in light gray. The NTD position uponbinding tRNA predicted by homology modeling is shown in dark gray. The CTD is also shown in dark gray. (b) Thesimulated SAXS profile of the rigid-body model (broken line) and the homology model (continuous line; calculated withouttRNA in the model) are shown fitted to the experimental SAXS data (circles). The better visual fit and lower χ2 value of thetRNA-free model shows that the SAXS data are able to clearly distinguish between which model exists in solution.


These electrostatic interactions along the inner faceof the NTD were speculated12 to be responsible forthe nonspecific RNA binding activity.29 This newconformation of the NTD is in agreement with thetRNA-bound conformation seen in the T. maritimaGatB structure. The hinge motion in our model isstrongly supported by our previous study demon-strating that mutations in conserved residues in thehinge region, connecting the helical and tail domainsof the NTD, significantly reduce tRNA binding.12 Inaddition, our modeling predicts that residues K29,K63, G64, T65, and D66 of the helical subdomain ofthe NTD make several contacts with the CTD, whichincludes residues P238 and M241 of Insertion 1.Since Insertion 1 was shown above to be exclusiveto eukaryotes, we speculate that this insertion mayprovide a means of communication between theNTD and CTD.The molecular dynamics simulation shows a

significant distortion of the tRNA molecule as a resultof the NTD binding to tRNA. At the start of thesimulation, base U73 is pointed away from the innercore of the enzyme, making no specific contacts withthe protein. Approximately 3 ns into the simulation,upon distortion of the tRNA molecule, this base flipsinwards toward the enzyme, resulting in contacts withLeu463. This conformational change in the tRNA,caused by binding of the NTD, results in these

previously unknown interactions between Leu463and U73. Our previous study reported that thepresence of the NTD across eukaryotes is stronglycorrelated with the existence of base U73 of theacceptor stem, immediately preceding the CCA tail.12

Furthermore, it was shown in E. coli GlnRS that anyalterations to base 73 led to substantial increases inKM for glutamine.17 Our molecular dynamics simula-tion showing these concerted conformationalchanges, combined with the phylogenetic correlationof the NTD and base U73 and the increase in KM forglutamine when mutating base 73 in E. coli GlnRS,may explain the result that deletion of the NTD in Gln4leads to increased KM for glutamine.12

Previously it was shown that GatB provides tRNAdiscrimination ability to GatCAB through the taildomain.30 This discrimination occurs through aninteraction with U20B of tRNAglu that is absent intRNAgln in prokaryotes. Analysis of tRNAgln andtRNAglu sequences in yeast reveals that base U20Bis present in both tRNA sequences, suggesting thatthis base does not participate in tRNA discriminationinteractions with Gln4. Our model of Gln4 bound totRNAgln reveals no evidence of significant interactionsbetween tRNAgln and the NTD that would otherwisebe negated by replacement with tRNAglu. This isconsistent with previous studies demonstrating non-specific RNA binding activity of the NTD.28,29

image of Fig.�5

Fig. 6. NTD undergoes confor-mational change after binding totRNA. (a) Plot of backbone RMSfluctuation as a function of residue.The RMS has been calculated asthe deviation from the starting struc-ture. (b) Structure of Gln4 before(blue) and after (red) moleculardynamics simulation. The tRNAmolecule has been removed forclarity.


Therefore, in contrast to the tRNA discriminationproperties of GatB, our model provides no evidencethat the Gln4 NTD makes any direct contacts withtRNAgln that would confer any additional capacity fordiscrimination against tRNAglu, suggesting that dis-crimination occurs via specific contacts with the CTD.

Conclusion

The structure of the CTD of Gln4 presented herewas shown to be highly similar to the E. coli GlnRSstructure. However, multiple insertions relative toE. coli GlnRS revealed insights into one structural

motif common to all eukaryotes and twomotifs specificto fungi. Since most eukaryotic GlnRS species lackthe bacterial unpairing loop that is proposed to play arole in tRNA discrimination, then another mechanismmust be employed to discriminate between tRNAglu

and tRNAgln in eukaryotic GlnRS species. Therefore,in eukaryotes, there may be a compensating mech-anism to discriminate between G1:C72 of tRNAgln

from U1:A72 of tRNAglu or, as in the case of non-discriminating archaeal GluRS,18 the first base pairdoes not play a significant role in tRNA discrimination.Our structure-based models of the first full-length

eukaryotic GlnRS with and without tRNAgln boundsuggest that CTD binding to tRNA results in a large

image of Fig.�6

Fig. 7. Comparison of NTD structure before and aftermolecular dynamics simulation. The position of the NTDbefore the simulation is shown in light gray and that afterthe simulation is shown in dark gray. The black continuousarrow shows the degree and direction of angular motioncalculated by DynDom.


conformational reorientation of the NTD allowing forinteractions between the NTD and the tRNA. Giventhe distinct increases in KM and KD for tRNAgln

following deletion of the NTD,10,12 the solution modelof full-length Gln4 presented here suggests that theNTDplays a direct role in tRNAbinding.Ourmoleculardynamics simulation revealed that the helical and tailsubdomains of the NTD undergo a hinge motion afterbinding to tRNA, allowing for tighter binding betweenthe NTD and tRNA. Our structural results andmodeling suggest the intriguing possibility that theNTD communicates with the CTD through Insertion 1,which is found in all eukaryotes. The absence of suchan interaction may explain the loss of the NTD inbacterial GlnRS evolution. In addition, since the NTDand the active site of Gln4 are too distant to interactdirectly and since deletion of the NTD also increasesKM for glutamine and ATP, it seems plausible that theeffects on glutamine andATPare due to the concertedconformational changes inGln4 that occur upon tRNAbinding as was observed in E. coli GlnRS.10,12,13,15

Further experimental studies will be required toconfirm these proposed interactions in eukaryoticGlnRS.

Materials and Methods

Protein expression and purification

To obtain highly purified Gln4 protein and its derivatives,we cloned open reading frames into the previouslydescribed LIC vectors BG2483 or BG2663 under PGAL1control31 as previously described12 and expressed them inyeast strain BCY123.32 To purify full-length Gln4 from a

Gln4-pt-tagged construct (QB721B), we performed large-scale growth, affinity purification on IgG Sepharose,removal of GST-3C protease, concentration of samples,and sizing on Superdex HiLoad 16/60 (GE Healthcare 17–1069, 10 mm × 300 mm bed dimension) as previouslydescribed.33 This purified Gln4 construct was used forcrystallographic and structural analyses.To ensure that full-length polypeptide was purified for

crystallography, we added an N-terminal Met-Ala-His6 tagat the N-terminus ofGLN4, during PCR amplification of theGLN4 gene using QB832ADFP and QB832ADRP primers(Supplementary Table 1). Full-length GLN4 was amplifiedwith QB1012ADFP and QB832ADRP.For crystallography of the full-length His6-Gln4 protein,

the His6-Gln4-pt was purified from strain QB832AD (21.6 Lat 6.5 OD/L) on 24 mL IgG Sepharose and eluted with64 mL 3C cleavage buffer.33 After elution from IgGSepharose and removal of GST-3C protease but prior tosizing, the sample containing the full-length Gln4 proteinwith the N-terminal His6 tag (QB832AD) was diluted with anequal volume of buffer T [25 mM Hepes (pH 7.5), 850 mMNaCl, 10% (v/v) glycerol, 2 mM β-mercaptoethanol (BME),and 2 mM phenylmethylsulfonyl fluoride], mixed with 3 mLprewashed Talon resin [washed in Wash buffer, 20 mMHepes (pH 7.5), 0.5 M NaCl, 5% (v/v) glycerol, and 2 mMBME], incubated for 1 h at 4 °C, followed by centrifugationat 2000 rpm for 2 min and removal of supernatant, afterwhich the Talon resin was washed once for 10 min with40 mL Wash buffer containing 0.5 M NaCl [20 mM Hepes(pH 7.5), 0.5 M NaCl, 5% (v/v) glycerol, and 2 mM BME],centrifuged at 2000 rpm for 2 min, followed by two morewashes of the resin with Wash buffer containing 1 M NaCl,then a wash with Wash buffer containing 0.5 M NaCl,followed by awashwithWash buffer containing 0.5 MNaCland 10 mM imidazole. The protein was eluted from theTalon resin with four sequential washes of the resin withWash buffer that contains 0.5 M NaCl containing 250 mMimidazole, each of which was mixed for 10 min prior to thelow-speed spin. Three fractions (elutions 1 and 2 as well asthe 10 mM imidazole wash) were combined and dilutedwith 75 mL of no-salt buffer to bring the NaCl to 0.2 M,followed by concentration to 5 mL using an Amicon(Millipore UFC901024), and loaded onto a Superdex 200sizing column as previously described.33

Crystallization

Initial crystallization conditions were identified using ahigh-throughput microbatch-under-oil method with a 1536-condition, incomplete-factorial-based screen.34 Conditionsthat produced crystals appearing suitable for optimizationwere prioritized according to their ease of cryoprotection35

and optimized using a drop volume ratio versus temper-ature technique.36 Crystals of full-length Gln4 wereprepared for diffraction by combining 3.5 μL of proteinsolution [13.9 mg/mL protein in 200 mM NaCl, 5% (v/v)glycerol, 2 mM DTT, 0.025% (w/v) NaN3, and 20 mMHepes buffer (pH 7.5)] with 2.0 μL of precipitant solution[50 mM NH4Br, 50 mM KC2H3O2, 100 mM Hepes(pH 7.5), and 20%(w/v) polyethylene glycol 20,000],incubated at 14 °C. The optimized 16:9 protein-to-precip-itant ratio and 14 °C temperature were determined from adrop volume ratio versus temperature technique. Crystalsappeared after 4 weeks.36

image of Fig.�7


Single crystal data collection and structure solution

Crystals of the full-length protein were harvested andcryoprotected, then shipped to Stanford Synchrotron Radi-ation Lightsource (SSRL). Single crystal X-ray data werecollected remotely on beamline 11-1.37 An initial excitationscan revealed the presence of zinc. Data were collected to2.15 Å, integratedwith XDS,38–40 and reducedwithScala,41

part of the CCP4 package.42 Initial molecular replacementwith PHENIX43 using an ~40% sequence homology E. coliGln-tRNA synthetase (PDB ID: 1GTS)14 failed, but acombined molecular replacement/single-wavelength anom-alous dispersion approach with the zinc signal wassuccessful. An iterative process of PHENIX refinementand manual model building through Coot44 was employedwith validation using MolProbity.45 The coordinates weredeposited as PDB ID: 4H3S. Data collection, processing,and refinement statistics are given inTable 1. TheN-terminalregion, residues 1–214, was unresolved in the electrondensity map. SDS-PAGE gels indicated that it was presentin the crystals. A similar procedure was followed todetermine the structure of the His tag purified protein.Western blot analysis again confirmed that the NTDresidues were present in the crystals. PyMOL46 was usedto generate symmetry mates, analyze solvent channels andcrystallographic packing, and produce images.

Bioinformatics

The DISOPRED2 prediction of protein disorder serverwas used to predict disordered residues.32 Primarysequences were taken from the UniProt database.47

Structures of GlnRS from E. coli (PDB ID: 1NYL) andD. radiodurans (PDB ID: 2HZ7) and GluRS from Metha-nobacterium thermoautotrophicus (PDB ID: 3AII) weretaken from the PDB.5,8,15,18 Sequence data and structuraldata were combined for use in the PROMALS3D multiplesequence and structure alignment server,48 and theresulting alignment was used to identify structural motifsdiscussed in the text. tRNA sequences were taken fromthe tRNAdb 2009 online database.49 BLAST (Basic LocalAlignment Search Tool) was used to calculate expectationvalues (E values) for comparing sequences.50,51

Small-angle X-ray scattering

Small-angle X-ray solution scattering data were collectedonbeamline 4-2 of theSSRL.52 Datawere collected from thefull-length protein at a wavelength of 1.3 Å for eightconsecutive 2-s exposures collected at five differentconcentrations ranging from 1.0 to 7.7 mg/mL. Data werecollected from the flow-through buffer of the final purificationcolumn [200 mM NaCl, 5% (v/v) glycerol, 2 mM DTT,0.025% (w/v) NaN3, and 20 mM Hepes buffer (pH 7.5)]and subtracted from the total protein solution scattering. Thedata were integrated with SasTool52 and then examinedwith PRIMUS.53 Analysis of eight consecutive time framesensured that no radiation damage tookplace over the courseof the experiment. The SAXS data for different proteinconcentrations were investigated for aggregation usingGuinier plots.21 Radius of gyration estimates were derivedby the Guinier approximation I(q) = I(0)exp(−q2Rg

2/3) with

qRg b 1.3 using the radius of gyration function of PRIMUSwhere q = 4πsinθ/λ. GNOM54 was used to compute the pairdistribution function, P(r), and to determine the maximumparticle dimension, Dmax. No evidence of concentrationdependence was seen when comparing all five concentra-tions. Chicken egg white lysozyme was used as a proteinstandard to estimate the molecular weight from I(0)extrapolated from the scattering curve using GNOM inreciprocal space. Porod–Debye analysis and Porod volumeestimations were performed using a pre-release version ofthe software SCÅTTER22 and confirmed with PRIMUS. Tenab initio shape reconstructions were generated byDAMMIF55 and averaged with DAMAVER.56 Ensemblemodeling was carried out with the ensemble optimizationmethod24 using default parameters. Fifty identical runswereperformed using 20 conformers in the ensemble selectedfrom among 10,000 random conformations of full-lengthGln4, while treating the NTD and CTD crystal structures asindependent rigid bodies and allowing the linker (residues188–214) to be flexible. Rigid-bodymodelingwas performedsimilarly using only one conformer in the ensemble, whichselected the best single conformation fromamong all 10,000random conformations.

Molecular dynamics

The initial model used for molecular dynamics simulationwas generated as described in the text. All structuralalignments were performed using the “fit” function ofPyMOL using carbon α atoms. Molecular dynamicssimulations were performed in GROMACS with theAMBER99SB force field.57,58 The initial model wassolvated using a cubic SPC/E water model59 and neutral-ized with ions prior to minimization via steepest descents.Distance restraints were added to keep the zinc ion inplace. The model was then equilibrated under an isother-mal–isochoric ensemble for 100 ps at 300 K followed byequilibration under an isothermal–isobaric ensemble for100 ps to ensure stability prior to the simulation. Simula-tions were then performed at the Center for ComputationalResources60 on 512 processors. Total simulation time was70 ns. The DynDom Protein Domain Motion Analysis Webserver27 was used to compare models. RMSD from thestarting structure was calculated for each frame of thetrajectory after fitting to the starting structure. RMSfluctuation was calculated as the deviation from the startingstructure after fitting to the starting structure.

Accession numbers

The coordinates and the structure factors have beendeposited in the PDB with accession number 4H3S.

Acknowledgements

Wewould like to thank the staff at SSRL beamline 4-2, Drs. Thomas Weiss, Tsutomu Matsui, and LesterCarter. We gratefully thank Dr. Robert Rambo at theAdvanced Light Source for access to a pre-release


version of SCÅTTER and for helpful discussions. Wewould also like to acknowledge Dr. Alan Grossfield atthe University of Rochester for helpful discussionsregarding molecular dynamics protocols and analysisand Cynthia Cornelius at the University at Buffalo'sCenter for Computational Resources for technicalassistance and advice. Portions of this research werecarried out at the Stanford Synchrotron RadiationLightsource, a national user facility operated byStanford University on behalf of the U.S. Departmentof Energy, Office of Basic Energy Sciences. The SSRLStructural Molecular Biology Program is supported bythe Department of Energy, Office of Biological andEnvironmentalResearch, andby theNational Institutesof Health, National Center for Research Resources,Biomedical Technology Program. Financial supportwas provided by the Defense Threat ReductionAgency (Grant HDTRA1-10-C-0057 to E.H.S.) andNational Institutes of Health (R01 GM100494).Dedication. We would like to dedicate this article in

memory of the late Dr. Hiro Tsuruta who providedvaluable guidance and instruction for the collectionand analysis of small-angle X-ray scattering data.

Supplementary Data

Supplementary data to this article can be foundonline at http://dx.doi.org/10.1016/j.jmb.2013.03.043

Received 7 November 2012;Received in revised form 25 February 2013;

Accepted 30 March 2013Available online 10 April 2013

Keywords:eukaryotic glutaminyl-tRNA synthetase;

structure;C-terminal domain;

small-angle X-ray scattering;molecular dynamics

Abbreviations used:NTD, N-terminal domain; CTD, C-terminal domain;

SAXS, small-angle X-ray scattering; PDB, Protein DataBank; NSD, normalized spatial discrepancy; BME,β-mercaptoethanol; SSRL, Stanford Synchrotron

Radiation Lightsource.

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