Toward understanding phosphoseryl-tRNACys

formation: The crystal structure of Methanococcusmaripaludis phosphoseryl-tRNA synthetaseSatwik Kamtekar*, Michael J. Hohn*, Hee-Sung Park*, Michael Schnitzbauer*, Anselm Sauerwald*, Dieter Soll*†,and Thomas A. Steitz*†‡§

Departments of *Molecular Biophysics and Biochemistry and †Chemistry and ‡Howard Hughes Medical Institute, Yale University, New Haven, CT 06520-8114

Contributed by Thomas A. Steitz, December 22, 2006 (sent for review December 16, 2006)

A number of archaeal organisms generate Cys-tRNACys in a two-step pathway, first charging phosphoserine (Sep) onto tRNACys andsubsequently converting it to Cys-tRNACys. We have determined, at3.2-Šresolution, the structure of the Methanococcus maripaludisphosphoseryl-tRNA synthetase (SepRS), which catalyzes the firststep of this pathway. The structure shows that SepRS is a class II,�4 synthetase whose quaternary structure arrangement ofsubunits closely resembles that of the heterotetrameric (��)2

phenylalanyl-tRNA synthetase (PheRS). Homology modeling of atRNA complex indicates that, in contrast to PheRS, a single mono-mer in the SepRS tetramer may recognize both the acceptorterminus and anticodon of a tRNA substrate. Using a complex withtungstate as a marker for the position of the phosphate moiety ofSep, we suggest that SepRS and PheRS bind their respectiveamino acid substrates in dissimilar orientations by using differentresidues.

cysteine � methanogen � phosphoserine � archaea �aminoacyl-tRNA synthetase

Understanding the pathways through which simple systemsevolve into more complex ones is a central concern in biology,

one that has led to interest in elucidating the history of theapparatus for the translation of the genetic code into proteinsequences. Aminoacyl tRNA synthetases (aaRSs), which chargetRNAs with the appropriate amino acids, provide a critical link inthis apparatus. They are a diverse group of enzymes with exquisitespecificity for both their amino acid and tRNA substrates (1). Manystudies have therefore attempted to reconstruct the evolutionaryhistories of these enzymes (1–5). Apart from the direct route toaa-tRNA catalyzed by the aaRSs, an indirect pathway involvingmisacylation and subsequent tRNA-dependent amino acid modi-fication operates in a number of cases, mainly for glutaminyl- andasparaginyl-tRNA formation (1, 6).

Although all aaRSs use a common two-step mechanism, firstactivating amino acids through the formation of aminoacyl–adenylate intermediates and then transferring the aminoacyl moi-eties onto the 3� termini of tRNAs (1, 7–9), they can be divided intotwo evolutionarily independent classes (10, 11). The catalyticdomains of class I synthetases contain Rossmann dinucleotide-binding folds (12, 13), whereas those of class II enzymes possessantiparallel � folds (11). The class II catalytic domains share threeidentifiable sequence motifs. Motif I consists of a helix, turn, and �strand and is typically part of an oligomeric interface, whereasmotifs II and III form part of the active site. Additional motifs,sometimes associated with amino acid-binding pockets, permit thefurther classification of synthetases with respect to their amino acidsubstrates (2, 3, 5, 9, 14).

Several methanogenic archaeal organisms, although encodingproteins that contain cysteine, lack a gene encoding canonicalcysteinyl-tRNA synthetase (CysRS) or have a dispensable formof this gene (3, 15, 16). This discrepancy has recently beenresolved by the discovery of an indirect pathway for the forma-

tion of Cys-tRNACys in these organisms (16). A class II syn-thetase in these methanogens charges tRNA with Sep to formphosphoseryl-tRNACys (Sep-tRNACys), which is subsequentlyconverted to Cys-tRNACys. This indirect pathway of Cys-tRNAformation is strikingly similar to the way eukaryotes and archaeasynthesize selenocysteinyl-tRNA; both cases involve a Sep-tRNA intermediate (16, 17). One of the reasons why this indirectpathway to Cys-tRNACys was not identified earlier was that genesencoding SepRS were initially, and in some databases continueto be, misannotated as � subunits of the heterotetrameric (��)2phenylalanyl tRNA synthetase (PheRS) because of the sequencesimilarity of these two synthetases.

Crystal structures of the PheRS from Thermus thermophilushave shown that PheRSs are unusually complex synthetases (18,19). With the notable exception of the monomeric, mitochon-drial Saccharomyces cerevisiae enzyme (20), they are heterotet-ramers. Their � subunits contain an N-terminal coiled-coil thatis involved in tRNA recognition, followed by a class II catalyticdomain. Their � subunits are much longer (785 aa long in T.thermophilus) and contain a catalytically inactive class II anti-parallel �-fold as well as domains involved in tRNA recognitionand editing (21–24). The sequence identity between the activeand inactive catalytic domains of the � and � chains of PheRSis only 15% on average (25); nevertheless, the structures of theseT. thermophilus domains are very similar, with an rmsd of 2.1 Åcalculated �166 C� pairs (18). There are two tRNA moleculesper heterotetramer in the cocrystal structure, with each tRNAmolecule contacting all four protein monomers. Thus the activesite, anticodon-binding domain and N-terminal coiled coil thatbind a tRNA molecule are each provided by separate polypep-tide chains (19).

Although the primary sequences of SepRSs show that theirclosest relatives are PheRSs, the two families of enzymes mustbe significantly different. Both PheRSs and SepRSs recognizethe different anticodons of their respective tRNAs (19, 26), andthe details of these interactions must therefore differ. Further-more, whereas in PheRS heterotetramers, the active sites aresupplied by � subunits and the anticodon binding domains by �subunits, SepRSs are encoded by single genes and contain onlyone type of polypeptide. Perhaps the most striking difference

Author contributions: S.K. designed research; S.K., M.J.H., H.-S.P., M.S., and A.S. performedresearch; S.K., M.J.H., H.-S.P., D.S., and T.A.S. analyzed data; and S.K. wrote the paper.

The authors declare no conflict of interest.

Abbreviations: Sep, phosphoserine; SepRS, phosphoseryl-tRNA synthetase; PheRS,phenylalanyl-tRNA synthetase; CysRS, cysteinyl-tRNA synthetase; AspRS, aspartyl-tRNAsynthetase; aaRSs, aminoacyl tRNA synthetases; NCS, noncrystallographic symmetry.

Date deposition: Coordinates and structure factors have been deposited in the Protein DataBank, www.pdb.org (PDB ID code 2ODR).

§To whom correspondence should be addressed. E-mail: eatherton@csb.yale.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0611504104/DC1.

© 2007 by The National Academy of Sciences of the USA

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between the two families of enzymes lies in the amino acids theyrecognize. Because phenylalanine and phosphoserine do notresemble each other in shape, hydrophobicity, or charge, the twofamilies of synthetases must have evolved very different aminoacid binding pockets. To address these issues, and to furtherunderstand the evolutionary relationship between PheRS andSepRS, we undertook structural and biochemical studies of theSepRS from M. maripaludis.

ResultsStructure Determination and Domain Organization of SepRS. We havedetermined the structure of the SepRS from M. maripaludis at 3.2Å resolution. Initial phases were obtained through multiple iso-morphous replacement by using tungstate and samarium deriva-tives. Preliminary model building into the resulting electron densitymap revealed the presence of a tetramer of SepRS within eachasymmetric unit. Taking advantage of this redundancy, the phases

N-terminal domain

C-terminal domain

Catalytic domain

Inserted helicaldomain

Pseudo two-foldsymmetry axes

Pseudo two-foldsymmetry axes

α SepRSβ PheRS

α SepRSβ PheRS

α SepRSα PheRS

α SepRSα PheRS

N-terminal domain

C-terminal domain

Catalytic domain

Inserted helicaldomain

Met 62

Thr 45

Motif 1Motif 2

MmSep 41 VFGKTHPVNDTIENLRQAYLRMGFEEYINPVIVDERDIYKQFGPEAMAVLDRCFYLAGLPRPDVGLSDEKISQIEKLGIKV 121MjSep 52 VYGKPHPVMETIERLRQAYLRMGFEEMINPVIVDEMEIYKQFGPEAMAVLDRCFYLAGLPRPDVGLGNEKVEIIKNLGIDI 132TtPhea 96 FSGGLHPITLMERELVEIFRALGYQAVEGPEVESEFFNFDA---[13]---DTFWLTG-------[15]------------ 156 TtPheb 491 P-YRKEQRLREVLSGLGFQEVYTYSFMDPEDARRF----[5]----RLLL----------[8]------------ 534

MmSep 122 SEHK-ESLQKILHGYKKGTLDGDDLVLEISNALEISSEMGLKILEDVFPEFKDLTAVSSKLTLRSHMTSGWFLTVSDLMNK 201MjSep 133 DEEKKERLREVLHLYKKGAIDGDDLVFEIAKALNVSNEMGLKVLETAFPEFKDLKPESTTLTLRSHMTSGWFITLSSLIKK 213TtPhea 172 -----------------------------------------------------------RLLLRTHTSPMQVRYMVAHT—- 191TtPheb 542 ------------------------------------------------------------AALRTHLFPGLVRVLKENL-- 560

MmSep 202 KPLPFKLFSIDRCFRREQKEDKSHLMTYHSASCAIAGEGV---DINDGKAIAEGLLSQFG--FTNFKFIPDEKKSKYYTPE 277MjSep 214 RKLPLKLFSIDRCFRREQREDRSHLMSYHSASCVVVGEDV---SVDDGKVVAEGLLAQFG--FTKFKFKPDEKKSKYYTPE 289TtPhea 192 --PPFRIVVPGRVFRFEQ-TDATHEAVFHQLEGLVVGEGI---AMAHLKGAIYELAQALFGPDSKVRFQP--VYFPFVE—- 263TtPheb 567 [6]RALLFEVGRVFRE---------REETHLAGLLFGEGV[11]YFLLKGYLEALFARLG---LAFRVEAQ--AFPFLHPG 638

MmSep 278 TQTEVYAYHPKLKEWLEVATFGVYSPVALSKYGI-DVPVMNLGLGVERLAMISGNFADVREMVYPQFYEH 346MjSep 290 TQTEVYAYHPKLGEWIEVATFGVYSPIALAKYNI-DVPVMNLGLGVERLAMIIYGYEDVRAMVYPQFYEY 358TtPhea 263 PGAQFAVWWPEGGKWLELGGAGMVHPKVFQAV[15]VTGFAFGLGVERLAMLRYGIPDIRY 334TtPheb 638 VSGRVLVE------GEEVGFLGALHPEIAQELEL--PPVHLFELRL 675

Motif 2

Motif 1

Motif 3

Motif 3

Catalytic domainsA B

C D

E

Fig. 1. The structure of M. maripaludis SepRS. (A) The synthetase is a tetramer with pseudo 222 symmetry and is shown in surface representation, and the colorof each monomer differs. The surface of one of the monomers is transparent, revealing a ribbon representation of its structure. (B) The catalytic domains of theSepRS tetramer shown in the same orientation as in A. Superimposed on this core are the two � and two � chains of the T. thermophilus PheRS (18). (C) A monomerof SepRS in the same orientation, colored by domain and motif. (D) Representative electron density for a helix in motif 1. To provide an unbiased view, the mapwas calculated by using phases derived without the use of the model; phases were produced by solvent-flattening and NCS-averaging heavy atom-derived phases.The amplitudes were sharpened by a B factor of 100 and the contour level set at 2� to reveal side-chain density. (E) A structure-based sequence alignment ofthe catalytic domains of M. maripaludis SepRS, M. jannaschii SepRS, and the � and �-chains of the T. thermophilus PheRS. The secondary structure elements ofthe M. maripaludis SepRS are indicated and colored as in C.

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were improved through multidomain noncrystallographic symme-try (NCS) averaging and solvent-flattening. Sharpening of theobserved amplitudes by a B factor of 100 yielded a map thatcontained interpretable density throughout much of the molecule(Fig. 1). Subsequent rounds of building and refinement led to amodel with a crystallographic R factor of 29.2% and Rfree of 30.6%(Table 1).

Each SepRS monomer can be divided into four domains andtheir connecting linker regions (Fig. 1). A small, N-terminal,helix–turn–helix domain is formed by residues 4 to 26. An extendedlinker containing residues 27 to 46 connect this domain with thecatalytic domain (residues 47 to 99 and 179 to 343). As expected fora class II synthetase, the catalytic domain is composed of asix-stranded antiparallel �-sheet surrounded by helices and flankedby an additional � strand. This domain contains the three motifscharacteristic of the class II synthetases: motif I is a strand and helixwhich typically forms part of a quaternary structure subunit inter-face (residues 47 to 70), motif II contains two antiparallel strandsconnected by a loop (residues 211 to 233) and is expected to beinvolved in ATP binding and tRNA recognition, whereas motif IIIis composed of a strand and helix that presumably also interactswith ATP (residues 315 to 330). Two short linkers connect thecatalytic domain to an inserted domain that contains a helicalbundle (residues 100 to 178, including the linker residues). Thisdomain is poorly ordered, and its electron density is most inter-pretable in unsharpened maps truncated at 5 Å resolution. A shortlinker from residues 344–348 connects the catalytic domain to aC-terminal domain, which contains mixed � and � secondarystructures. Although some of the sequence of this domain has beenfitted to the electron density, in general, sequence register has notbeen established because of disorder. Much of this region, as wellas the sequence corresponding to the inserted helical bundledomain, have therefore been either omitted or traced as polyala-nine chains in the final model.

Quaternary Structure. The structure of SepRS establishes that it isa highly interdigitated, homotetrameric enzyme consisting of a coreand peripheral regions (Fig. 1). The core of this �4 assembly consistsof catalytic domains along with residues 30 to 46 that link it to theN-terminal domain. The N-terminal, inserted, and C-terminaldomains decorate the periphery of this core region (Fig. 1). Theinteractions between the core and peripheral domains bury surfaceareas that range from 360 to 900 Å2 when calculated per domain.Because some of the domains contain stretches of sequence thathave currently been modeled as polyalanine or are incompletelytraced, these values are a lower estimate. Nevertheless, theirrelatively small sizes suggest that the peripheral domains may beable to reorient and be positioned differently upon formation of thecomplex between tRNA and synthetase. Interestingly, all of theinteractions between peripheral domains and the core region occurin trans, that is, the peripheral domain in a given monomer onlyforms interfaces with the core domain of a different monomer. Thisfeature interlinks the monomers within the �4 particle. Overall, theassembly possesses approximate 222 point group symmetry, withthe core region obeying the symmetry more strictly than theperipheral domains.

The structure of the core of the M. maripaludis SepRS �4tetramer superimposes well on that of the core of the (��)2heterotetramer of T. thermophilus PheRS (Fig. 1). This superposi-tion simultaneously overlays the four catalytic domains of theSepRS tetramer with the two catalytic domains from the � chainsand the two inactive catalytic-like domains from the � chains ofPheRS. The rmsd between 525 corresponding C� atoms in the twoassemblies is 2.2 Å. In both cases the tetramer interface is composedof similar secondary structure elements (Fig. 1). This similarity inquaternary structure raises the possibility that the common ances-tor of PheRS and SepRS may have shared a similar homotetramericarrangement.

The peripheral domains of PheRS and SepRS share fewersimilarities than their core regions. Although the � subunits of bothenzymes contain an N-terminal helix–turn–helix, the helices inSepRS are less than half the length of those in PheRS. Furthermore,PheRS does not have a sequence insertion corresponding to theinserted domain of SepRS. Indeed, the only other synthetase ofknown structure that has a domain, albeit a smaller one, inserted inthe same region of the catalytic domain as SepRS is Methanosarcinabarkeri SerRS (27). Finally, database searches for similar structuresby using the DALI server (28) have failed to find significantsimilarities between the structure of the C-terminal domain ofSepRS and structures deposited in the protein data bank. This maysuggest that this domain adopts a novel fold, or alternatively may bea consequence of the incomplete modeling of this domain ofSepRS.

The Active Site. Incubation of the crystals of SepRS in solutionscontaining phosphoserine severely degraded the quality of theirdiffraction. The active site of SepRS was therefore analyzed in threeways: we compared it with the active site of PheRS, examined thebinding site of the phosphate mimic, WO4, and made active sitemutations (Fig. 2; Table 2).

Superposition of the structures of the active sites of M. mari-paludis SepRS and T. thermophilus PheRS revealed the location ofa number of conserved residues [Fig. 2B; and see supportinginformation (SI) Fig. 4]. In the structures of T. thermophilus PheRSbound to phenylalanyl-adenylate, or a nonhydrolyzable analog (29,30), the side chains of some of these conserved residues contact theadenylate base (F216 and E218), adenylate phosphate (R204), andcarbonyl (R204). Because these residues are conserved, it is plau-sible that they mediate contacts in SepRS similar to those observedin PheRS.

In contrast, the residues in the � chain of PheRS that interact withthe phenylalanine side chain are not conserved. In particular, F258,F260, V261, and A314, which define a hydrophobic pocket for the

Table 1. Crystallographic statistics

Measurement Value

Space group P212121

Resolution, Å 50.0–3.2Rmerge,* % 5.3 (91)I/�† 24.9 (1.1)Completeness, % 98.8 (94.4)Unique reflections 52,480Redundancy 3.7Copies in AU 4rmsd bond length, Å 0.008rmsd bond angle, ° 1.04Rcryst,‡ % 29.2Rfree,‡ % 30.6PDB ID code 2ODRPhasing power§ (acentric)

Sodium tungstate 1.6 (50–4.5 Å)Samarium acetate 1.1 (50–4.5 Å)

Overall figure of meritHeavy atom 0.52 (50–4.5 Å)Density modified 0.73 (50–3.2 Å)

*Rmerge is ¥j Ij � �I� , where Ij is the intensity of an individual reflection, and �I�is the mean intensity for multiply recorded reflections. The values in paren-theses are for the high-resolution shell (3.31–3.2 Å).

†I/� is the mean of intensity divided by the standard deviation.‡Rcryst is ¥�Fo � Fc�/¥ Fo , where Fo is an observed amplitude and Fc a calculatedamplitude; Rfree is the same statistic calculated over a subset of the data thathas not been used for refinement.

§Phasing power is the rms isomorphous difference divided by the rms lack ofclosure.

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phenyl side chain in T. thermophilus PheRS, correspond to residuesS271, Y273, Y274, and N317, respectively, in SepRS. As a conse-quence of these differences in sequence, the volume of the corre-sponding pocket is substantially reduced in size and is hydrophilicin SepRS (Fig. 2C). This structural observation accords withprevious biochemical results showing that SepRS does not activateor charge phenylalanine (16).

An additional set of differences permit SepRS to bind WO4 in afashion that PheRS could not (Fig. 2D). Two of the SepRS residuesthat coordinate WO4 are S231 and S233 and they correspond toQ218 and E220 respectively in T. thermophilus PheRS. Stericclashes, and in the case of E220, electrostatic repulsion, precludefavorable interactions between these PheRS residues and WO4.Furthermore, another residue that coordinates WO4 in SepRS,N317, is an alanine in PheRS, a residue that cannot.

Nine point mutations made in the active site help identify regionsthat may be directly involved in contacting the phosphoseryl-adenylate substrate of SepRS (Fig. 2A; Table 2). Mutation of eitherK272 or S271 lead to minor effects on catalytic activity, making it

unlikely that they directly contact substrate. In contrast, mutationsof residues H186, S231, S233, or N317 to alanine or Y273 tophenylalanine reduce the kcat/Km by �100-fold. Of these fiveresidues, only H186 is conserved in PheRS (T. thermophilus H178)where it is proximal to the �-amino moiety of phenylananine. Thesefive residues considered together, form a plausible binding site forphosphoserine, consistent with the role that some of them may playin binding WO4.

DiscussionThe results presented above have provided information on thearrangement of the active site of M. maripaludis SepRS, as well asits tertiary and quaternary structure. To further understand itsmechanism, we have used these structural results as a basis forconstructing homology models of the SepRS bound to tRNA andto the aminoacyl-adenylate.

Homology Modeling of Interactions Between Synthetase and tRNA.The tRNA from the structure of T. thermophilus PheRS complexedwith tRNA can be homology modeled onto the structure of SepRS

A

C

E F

D

B

Fig. 2. The active site of M. maripaludis SepRS compared with that of T. thermophilus PheRS (PDB ID code 1JJC) (29). (A) The catalytic domain of one monomerof SepRS (cyan) is shown superposed on a catalytic domain of PheRS (gray). This superposition is used in all images. Residues mutated in SepRS are shown in stickrepresentation. Residue labels in all images are colored to indicate the severity of the mutant phenotype, and corresponding residue numbers in PheRS are shownin brackets. (B) Three T. thermophilus PheRS residues that interact with the AMP moiety are shown in gray. The corresponding residues in M. maripaludis SepRSare conserved (green). (C) Four T. thermophilus PheRS residues that form a binding pocket for the phenylalanyl side chain are shown in gray. The correspondingresidues in M. maripaludis SepRS (green) are incompatible with the binding of phenylalanine. (D) Residues in M. maripaludis SepRS that may interact with thephosphate moiety of phosphoserine. Difference electron density, caused by incubating a crystal with sodium tungstate and contoured at 6�, is shown as a meshand superimposed on the structure both here and in F. (E and F) Side-by-side comparison of the interactions of T. thermophilus PheRS with phenylalanyl-adenylateand M. maripaludis SepRS with modeled phosphoseryl-adenylate. The side-chain conformations of M. maripaludis SepRS residues H186, T188, and R216 havebeen altered from the apo structure to accommodate the phosphoseryl moiety. The phosphoseryl phosphate group occupies a hydrophilic pocket, and its positionoverlaps the tungstate difference electron density.

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by using the superposition of the catalytic domain folds shown inFig. 1. The phosphodiester backbone of the tRNA modeled in thisfashion clashes with the SepRS main chain in two locations (Fig. 3).First, Gua1, Cyt74, and Cyt75 clash with the inserted domain.Second, the anticodon stem clashes with the C-terminal domain ofthe synthetase. One way to relieve these clashes would be throughadjustment of the position of the modeled tRNA. Such adjustmentsare within the range of accuracy expected for a homology modeledcomplex. For example, tRNA does not clash with the inserteddomain of SepRS if the homology modeling is based on the S.cerevisiae AspRS:tRNA structure (AspRS) (31) instead of the T.thermophilus PheRS:tRNA structure (although using the AspRSstructure does increase the number of clashes between SepRS andthe modeled anticodon stem). Alternatively, the orientations of theinserted and C-terminal domains within the SepRS tetramer mightchange upon binding tRNA. Such movements seem particularlylikely for the inserted domains because they are very poorly orderedin our current structure, and thus appear not to be locked in placein the absence of tRNA.

The homology model is consistent with mutational data that wereused to determine the identity elements of M. jannaschii tRNACys

(26). In particular, both the model and the data support theproposal that SepRS contacts both the acceptor and anticodon endsof the tRNA. Furthermore, the mutational studies indicate that thechanges G15A or C20U have only moderate effects on SepRSactivity, consistent with the observation that they are far from theprotein:tRNA interface in the homology model.

The N-terminal coiled-coil domain of SepRS does not interactwith the homology modeled tRNA, but could do so with relativelysmall movements. Because this domain abuts the back of the activesite, such a movement might link tRNA binding with changes at theactive site. If the conformational changes within this domain upontRNA binding are modest, it cannot interact with tRNACys in theextensive fashion that the much longer N-terminal coiled-coil of the� subunit of PheRS does with tRNAPhe. Alternately, the N-terminaldomain of SepRS might have more substantial contacts with tRNAif the binding of nucleic acid triggers the turn in this domain toassume a helical conformation. Secondary structure predictionalgorithms support such a possibility because they strongly score theturn as a helix.

Examination of the homology model suggests two further ways inwhich interactions of SepRS with tRNACys might differ from thoseof PheRS with tRNAPhe. First, the inserted domain of SepRS, whichhas no counterpart in PheRS, is poised to interact with tRNA.Second, in the model, the same monomer of SepRS that binds theacceptor end of a tRNA molecule in its active site, interacts with thetRNA anticodon through its C-terminal domain. This cis interac-tion is in marked contrast with PheRS, where � subunits provide theactive sites, and � subunits the anticodon binding domains.

Modeling of Phosphoseryl-Adenylate in the Active Site of SepRS. Inmodeling phosphoseryl-adenylate in the active site of SepRS, it isuseful to consider the adenylate and aminoacyl moieties separately.The residues in T. thermophilus PheRS that bind the adenylatemoiety of phenylalanyl-adenylate are conserved in SepRSs (Fig.2B; supplementary Fig. 1). Thus importing the adenylate moiety byusing a simple superposition of the active site of a PheRS cocrystalstructure provides a plausible model for SepRS interactions withthe adenylate.

The phosphoseryl moiety, by contrast, cannot be modeled byusing conserved interactions. In T. thermophilus PheRS, a recog-nition loop containing F258, F260, and V261 is involved in bindingthe phenylalanine side chain. The corresponding loop in SepRS ismore hydrophilic, and it had previously been proposed that residuesfrom this loop might bind phosphoserine (25). In particular, it wassuggested that M. jannaschii residues K281, S283, and K284 (K269,S271, and K272 respectively in M. maripaludis) might interactdirectly with the phosphate moiety. The functional consequences ofmutation of the M. maripaludis SepRS (Fig. 2; Table 2) stronglyargue against this suggestion. Furthermore, attempts to model theside chain in this region generate steric clashes because SepRS doesnot contain a pocket large enough to accommodate the phosphateat this location (Fig. 2; and see SI Fig. 4).

Tungstate, which has been previously used as a structural mimicof phosphate and thus is a marker for phosphate binding sites (32),provides an experimental constraint with which to model theposition of the side chain of phosphoserine. Structural superposi-tion of a catalytic domain of SepRS with a complex of S. cerevisiaeAspRS bound to tRNA and ATP (33) indicates that the tungstateis a marker for the position of a phosphate moiety in phosphoserinerather than one of the phosphates of an ATP substrate. ATP in theactive site of class II synthetases adopts a bent conformation (9, 33)and consequently, the distance between the tungsten and the �, �,and � phosphorous atoms of the ATP from the superposed complexand the tungsten are 6, 9, and 11 Å, respectively. The superpositionof the two synthetases aligns R324 in M. maripaludis SepRS withR531 in S. cerevisiae AspRS, a residue that interacts with the �phosphate of ATP. As expected, this indicates that SepRS alsobinds ATP in a bent conformation.

Fig. 3. tRNAPhe homology modeled onto the structure of SepRS by using thesuperposition of the catalytic domains of M. maripaludis SepRS and T. ther-mophilus PheRS (19) illustrated in Fig. 1B. The tRNAs are shown as white/graysurfaces; each polypeptide chain of SepRS is colored differently, and interac-tions between SepRS and one of the tRNAs are labeled.

Table 2. Kinetic constants of phosphoserine activation by M.maripaludis SepRS mutants, measured by ATP–PPi exchange

SepRS Km (�M) kcat (sec�1) rel kcat/Km

Wild type 72.43 � 1.97 3.22 � 0.03 1H186A 737.92 � 34.53 0.156 � 0.004 0.0048S231A N.D. N.D. 0.0003*S233A N.D. N.D. 0.0003*K269A 127.67 � 28.60 0.50 � 0.038 0.089S271A 67.65 � 17.44 2.20 � 0.129 0.74K272A 80.67 � 20.03 0.79 � 0.06 0.22Y273F 343.46 � 74.20 0.01 � 0.001 0.007Y274F 287.93 � 81.53 1.95 � 0.29 0.15N317A N.D. N.D. �0.0001*

N.D., not determined.*The catalytic efficiency (kcat/KM) for this mutant was an approximation undersub-KM concentration of substrate.

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A model of phosphoseryl-adenylate in the active site of SepRS,shown in Fig. 2F, is consistent with structural and mutational data.The orientation of the adenylate monophosphate moiety, as well asthe protein interactions it makes, are very similar to those of theadenylate monophosphate moiety in a PheRS structure (29). Incontrast, to avoid steric clashes and to position the serine linkedphosphate at the location of the bound tungstate, we conclude thatthe orientation of the cognate aminoacyl group bound to SepRSdiffers from that bound to PheRS.

ConclusionsSepRS and PheRS differ in substantial ways. PheRSs, with theexception of the mitochondrial S. cerevisiae enzyme (20), areheterotetrameric, whereas SepRSs are homotetramers. Further-more, similarities between the two families of synthetase do notappear to extend beyond their core catalytic domains. Perhapsmost significantly, homology modeling of the SepRS substratecomplex suggests that these homologous synthetases interactwith both their tRNA and amino acid substrates in ways thatdiffer. For example, the catalytic active site in PheRS is providedby the � subunit and tRNA anticodon recognition is mediatedin trans by the � subunit, whereas in SepRS one monomer mayprovide both the anticodon recognition domain and the activesite. The amino acid binding sites of the two families of syn-thetases also appear to be spatially distinct.

Despite these differences, the two families of synthetases share anumber of similarities. The catalytic domains of SepRS and PheRSare structurally more similar than the catalytic and catalytic-likedomains of the � and � chains of PheRS (28). The SepRS andPheRS families also share a distinct, superimposable quaternarystructure. These similarities are all the more remarkable becausethe divergence of the two families may predate the last universalcommon ancestral state (25).

Materials and MethodsWild-Type and Mutant SepRS Gene Constructs and Enzyme Purifica-tion. Mutants were constructed by using a QuikChange mutagen-esis kit (Stratagene, La Jolla, CA) and a previously cloned M.maripaludis SepRS gene in a pET15b expression vector as atemplate (26). Mutations were confirmed by sequencing. Bothwild-type and mutant His6-tagged M. maripaludis SepRSs werepurified to apparent homogeneity as described (26). Purified pro-tein was dialyzed against a buffer containing 20 mM Hepes-KOHpH 8.0, 40 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, and 10%glycerol.

Crystallization. Before crystallization, the protein was dialyzed intoa buffer containing 20 mM Na Hepes (pH 7.5), 5 mM DTT, and

5% glycerol. Crystals were grown by hanging-drop vapor diffusion,mixing equal volumes of protein (6–22 mg/ml) with well solutioncontaining 4–6% PEG 35000, 5 mM DTT, 100 mM Na MES (pH6.5), and 5% glycerol. They appeared overnight, and reacheddimensions of up to 0.2 � 0.2 � 0.5 mm. The orthorhombic crystalswere stabilized in 5–8% PEG 35000, 5 mM DTT, 50 mM Na MES(pH 6.5), and 5% glycerol and cryoprotected by increasing the finalglycerol concentration to 30% in three steps of 5 min each beforeflash-freezing in liquid propane.

Structure Solution and Refinement. Native crystals diffracted at bestto 3.2 Å resolution and contained four monomers per asymmetricunit (Table 1). Initial phases were obtained by using crystalsincubated with either 1 mM NaWO4 for 2 hours or with 1 mM Smacetate for 30 minutes. These multiple isomorphous replacementphases were calculated by using the program SOLVE (34), andimproved through the use of multidomain noncrystallographicsymmetry averaging as implemented in the programs DM (35) andRESOLVE (36). Sharpening of the map by a B factor of 100produced electron density that was interpretable over much ofsynthetase. Cycles of manual building that use the program COOT(37) and refinement with the program REFMAC (38) led to amodel with a crystallographic R factor of 29.2% (Rfree of 30.6%)(Table 1). Figures were made by using Pymol (www.pymol.org).

Characterization of SepRS Mutants. ATP-[32P]PPi exchange reactionassays were carried out at 37°C in 200 �l of buffer containing 50 mMHepes-KOH (pH 8.0), 100 mM MgCl2, 5 mM DTT, 1 mM KF, 4mM ATP, 2 mM 32PPi (5 cpm/pmol), 20–400 �M O-phosopho-serine, and 0.25 �M SepRS. Forty-microliter aliquots of the reac-tion were removed periodically, mixed with activated charcoal (200�l of 1% (wt/vol) Norit in 0.4 M sodium pyrophosphate solutionwith 15% (vol/vol) perchloric acid), and filtered on GF/C fiberglassfilter disks (Whatman, Clifton, NJ). Filters were washed with 15 mlof H2O and 7 ml of ethanol, dried, and liquid scintillation-counted.Kinetic parameters (KM and kcat) were determined from thecorresponding Eadie–Hofstee plots.

We thank the staff at National Synchrotron Light Source beamlines X25 andX29, at APS beamlines 19-ID and NECAT24-ID, and at Advanced LightSource beamlines 8.2.1 and 8.2.2; members of the T.A.S. laboratory for helpwith data collection and useful discussions; and the staff of the Center forStructural Biology core facility, especially Michael Strickler, at Yale. Thiswork was funded by National Institute of General Medical Sciences GrantsGM 22778 (to T.A.S.) and GM 22854 (to D.S.), Department of EnergyGrant DE-FG02-98ER20311 (to D.S.), National Science Foundation GrantMCB-0645283, and a Feodor Lynen Postdoctoral Fellowship of the Alex-ander von Humboldt Stiftung (to M.J.H.).

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