Structures of Tryptophanyl-tRNA Synthetase II from Deinococcus radiodurans Bound to ATP and Tryptophan INSIGHT INTO SUBUNIT COOPERATIVITY AND DOMAIN MOTIONS LINKED TO CATALYSIS*

An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radi-ation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. Crystal structures of drTrpRS II, empty of ligands or bound to either Trp or ATP, reveal that drTrpRS II has an overall structure similar to standard bacterial TrpRSs but undergoes smaller amplitude motions of the helical tRNA anti-codon binding (TAB) domain on binding substrates. TAB domain loop conformations that more closely resemble those of human TrpRS than those of Bacillus stearothermophilus TrpRS (bsTrpRS) indicate different modes of tRNA recognition by subclasses of bacterial TrpRSs. A compact state of drTrpRS II binds ATP, from which only minimal TAB domain movement is necessary to bring nucleotide in contact with Trp. However, the signature KMSKS loop of class I synthetases does not completely engage the ATP phosphates, and the adenine ring is not well ordered in the absence of Trp. Thus, progression of the KMSKS loop to a high energy conformation that stages acyl-adenylation requires binding of both substrates. In an asymmetric drTrpRS II dimer, the closed subunit binds ATP, whereas the open subunit binds Trp. A crystallographically symmetric dimer binds no ligands. Half-site reactivity

The most critical molecular recognition events in the translation of the genetic code involve the error-free attachment of 21 amino acids to tRNAs that bear the appropriate anticodon triplets (1)(2)(3). These reactions are catalyzed by aminoacyl-tRNA synthetases (AARSs), 1 which are divided into two classes (I and II) based on their structures and reactivities (4). Class I synthetases attach amino acids to the 2Ј-hydroxyl group of the 3Ј-terminal adenosine of tRNA and comprise a catalytic Rossmann fold (RF) domain and a helical tRNA anticodon binding domain (TAB) (5). Two signature sequence motifs (KMSKS and (T/H)IGN) that participate in ATP binding typify class Ic synthetases (TrpRS, TyrRS, and PheRS). Some class I synthetases also incorporate an insertion domain (CP1) and participate in additional cellular functions such as RNA splicing, tRNA processing (6) RNA trafficking, and apoptosis (7).
Although most AARSs are usually expressed from single copy genes to avoid the proliferation of synthetase mutations that could corrupt the genetic code, two copies of some synthetases are found in select bacteria and archea. In particular, Deinococcus radiodurans contains two tryptophanyl-tRNA synthetases (TrpRSs). Whereas D. radiodurans TrpRS (drTrpRS) I has 40% sequence identity to most single copy bacterial TrpRSs, drTrpRS II is more divergent, with only 28% sequence identity (8). We have recently shown that drTrpRS II interacts with D. radiodurans nitric oxide synthase (8,9) and can charge tRNA with Trp and 4-nitrotryptophan, a specific nitration product of D. radiodurans nitric oxide synthase. drTrpRS II is induced during responses to DNA damage. It contains an unusual 30-residue extension, uncharacteristic of any other known prokaryotic TrpRSs (8) but homologous to the N terminus of the stationary phase stress-response protein, SurE (10).
Extensive crystallographic work by Carter and colleagues (11)(12)(13) has established TrpRS from Bacillus stearothermophilus (bsTrpRS) as a paradigm for understanding the structural basis of enzymatic activity for class Ic AARSs. Crystal structures of bsTrpRS in three distinct conformations (open, closed, and closed pretransition state (pre-TS)) provide snapshots of important states in the catalytic cycle (12)(13)(14). An open conformation binds either ATP or Trp in a kinetic scheme described as "random bi-bi" (15,16). Favorable binding interactions provided by the second substrate overcome the stability of the open state and result in domain closure. This high energy closed conformation (inferred in the closely related TyrRS from kinetic studies (17,18)) engages the conserved KMSKS loop of the TAB domain with the ATP pyrophosphate group and brings the Trp carboxylate in close proximity of the ATP ␣-phosphate (12). The strain generated in the pre-TS drives formation of the acyl-adenylate by destabilizing the enzyme-substrate ground state. More recently, the crystal structure of ligand-free human TrpRS (hTrpRS) has revealed one conformation that resembles the bsTrpRS closed form (19).
Substrate binding and catalytic properties of prokaryotic and eukaryotic TrpRSs share some common features that complement similarity in their overall structures (20). Both the Escherichia coli and bovine TrpRSs bind two Trps per dimer. However, E. coli TrpRS binds both Trps with nearly identical affinity, whereas bovine TrpRS exhibits anticooperative binding (20). Both the E. coli and bovine TrpRS exhibit no cooperativity in ATP binding. This contrasts with ATP binding by bsTrpRS, where the second molecule of ATP binds with a drastically reduced affinity compared with the first (12). However, since all the structures of bsTrpRS are highly symmetric (most due to crystallographic symmetry), little information is available about the mechanism of intersubunit communication in TrpRSs.
The mode of tRNA recognition by bsTrpRS has largely been inferred from the structures of other class I synthetases (such as TyrRS) in complex with tRNA (13,19). These structures indicate that one tRNA binds both subunits of the dimer simultaneously (21)(22)(23). The TAB domain of one subunit binds the anticodon, whereas the RF domain of the adjacent subunit catalyzes aminoacylation (12,23). It was proposed, based on structural homology of bsTrpRS to TyrRS, that adjacent loops comprising residues 219 -226 and residues 259 -266 of the TAB domain recognize the tRNA anticodon (13). Mutational studies in Bacillus subtilis TrpRS indicate that a region close to the first TAB domain loop (residues 233-237 in bsTrpRS) are involved in binding the anticodon (24), whereas an RF domain helix (␣ 6 , residues 106 -120, bsTrpRS numbering) recognizes the tRNA acceptor arm.
Tryptophan recognition by bsTrpRS and drTrpRS II differs significantly. Our recent crystal structures of drTrpRS II bound to Trp (Protein Data Bank code 1YI8) or 5-hydroxy-Trp (Protein Data Bank code 1YIA) show that the presence of several residue substitutions (conserved by a subset of TrpRS IIs) alters orientation of the indole ring in the active center and thereby allows binding of Trp moieties derivatized at the 4-and 5-positions (25).
We now report the 2.4 Å crystal structures of drTrpRS II free and in complex with ATP and compare these structures with the Trp-bound enzyme. These structures reveal that Trp and ATP bind to the "closed" and "open" conformers of drTrpRS II, respectively. However, the closed and open conformations of drTrpRS II differ only modestly in TAB domain orientation, which more closely resembles that of hTrpRS than bsTrpRS. Furthermore, no large motions are required for the system to progress to a state competent for adenyl Trp formation. Also, in contrast to bsTrpRS, the conformations that bind substrates involve asymmetric dimers, in which only one subunit occupies ligand. Distinct structural changes propagate effects of ligand binding through the dimer interface to the adjacent subunit. These results explain subunit communication in TrpRS II and indicate that not all TrpRSs require large domain motions to couple protein conformational energy to ligand binding and catalysis.

MATERIALS AND METHODS
Crystallization, Data Collection, and Structure Determination of drTrpRS II-TrpRS II was prepared as previously described (20) and concentrated to 40 mg/ml in 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM Mg-ATP. Diffraction quality crystals were obtained by mixing 2 l of the protein solution described above with 2 l of well solution composed of 22% polyethylene glycol 4000, 0.2 M diammonium hydrogen phosphate. The protein drop was equilibrated against 500 l of well solution by vapor diffusion in a hanging drop setup at 25°C. Although 10 mM Mg-ATP was present at all steps of purification and crystallization, crystals were soaked in stabilization buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 22% polyethylene glycol 4000, 0.2 M ammonium phosphate) and 10 mM Mg-ATP 2 h prior to data collection. Higher concentrations of Mg-ATP were precluded by the crystallization of the nucleotide itself in the polyethylene glycol/phosphate buffer where the crystals are stable. Longer soaking times (4, 12, and 16 h) reduced the occupancy of ATP bound to TrpRS II in the crystals. Crystals were briefly soaked in cryoprotectant (22% polyethylene glycol 4000, 0.2 M diammonium hydrogen phosphate, 10 mM Mg-ATP, 10% methyl pentanediol) and flash frozen in liquid nitrogen prior to data collection. TrpRS II crystals diffracted to 2.4 Å resolution on beamline F1 of Cornell High Energy Synchrotron Source at 100 K. Data were integrated and scaled with HKL2000 (26). The resulting structure of drTrpRS II had one of three subunits bound to ATP. Initial phases were determined by molecular replacement with AMoRe (27) (correlation coefficient ϭ 0.57, initial R-factor ϭ 40.1%) with the previously reported structure of drTrpRS II as the model (25). Although these crystals belong to the same space group (C2) and have the same overall molecular packing arrangement as those of the previously reported structure (25), small changes in packing and cell dimensions required the use of molecular replacement. (Rigid body refinement with the coordinates from the Trp-bound structure would not converge to reasonably low R-factors.) Lack of isomorphism among different TrpRS II crystals was highly variable, and probably exacerbated by the flash freezing in liquid nitrogen required for synchrotron data collection. As a result, additional data sets were collected for crystals that were not soaked with nucleotide so that direct comparisons could be made to the ATP-containing crystals with difference Fourier maps. Diffraction data and refinement statistics for the most isomorphous non-ATP-containing TrpRS II structure is also reported in Table I. Subsequent model building and refinement with CNS (28) produced the final models for the two structures (Table I).
Isothermal Titration Calorimetry-BCA assay (Pierce) and RC/DC assay (Bio-Rad) were used with bovine serum albumin and cytochrome c standards to determine the total mass concentration (mg/ml) of TrpRS II (molecular mass of 38,465 Da). Calorimetric measurements were carried out using a VP-isothermal titration calorimeter (MicroCal) at temperatures at 25°C. Prior to titration with Trp, concentrated drTrpRS II samples were dialyzed extensively against gel filtration buffer (50 mM Tris (pH 7.5), 150 mM NaCl).

RESULTS
Overall Structure-The crystal structure of drTrpRS II resembles known structures of B. stearothermophilus TrpRS (bsTrpRS) and human TrpRS (hTrpRS), with exceptions of striking differences within the tRNA anti-codon binding (TAB) domain and the Trp-binding pocket. Like other TrpRSs, drTrpRS II forms an obligate dimer (Fig. 1, A and B). Two different dimers compose the crystals, but only one and onehalf dimers compose the asymmetric unit. Hence, in the first dimer, the two subunits (A and AЈ) are identical by crystallographic 2-fold symmetry, whereas in the second, the two subunits (B and C) are pseudosymmetric because of changes in relative domain orientations and loop conformations. The three crystallographically unique drTrpRS II subunits have distinctly different recognition properties with respect to substrates.
Two domains comprise the drTrpRS II subunit: a large Nterminal, RF domain (residues 21-202 and 317-351), which has the characteristic ␣␤␣ three-layered sandwich of dinucleotide-binding proteins, and a smaller, helical, TAB domain (residues 208 -307) (Fig. 1, A and B). The RF and TAB domains connect through two hinge regions that comprise an extended polypeptide segment (residues 203-207) and the junction between two long C-terminal helices (␣ 14 and ␣ 15 ) that bracket both domains (Fig. 2). Movement about the hinges results in three different TAB domain orientations for the three molecules in the asymmetric unit (Fig. 3).
Whereas the subunits of bsTrpRS show only subtle asymmetry in all known crystal forms and hTrpRS exclusively forms a crystallographic dimer, the B and C subunits of drTrpRS II are clearly not structurally equivalent (Fig. 3). Whereas the A and  (Fig. 1D).
The TAB Domain of drTrpRS II and hTrpRS Have Similar Structures and Orientations-Superposition of TAB domains of drTrpRS II and bsTrpRS I reveals striking differences in the loop regions that are implicated in tRNA Trp anti-codon recognition. Although three of the four helices adopt conformations similar to those of bsTrpRS, helix ␣ 12 tilts to bring the preceding ␣ 11 -to-␣ 12 loop closer to the loop connecting ␣ 13 and ␣ 14 (Figs. 2 and 4). The two loops interact with each other through a main-chain hydrogen bond between Gly 285 and Ala 249 (Fig.  4A). These peripheral TAB domain loops have very different conformations from those found in bsTrpRS (Fig. 4A), where the equivalent ␣ 11 -to-␣ 12 loop extends toward the active center cleft and does not interact with the ␣ 13 -to-␣ 14 loop. The equivalent loop regions of hTrpRS resemble those of drTrpRS II in that they also retract from the interdomain cleft (Fig. 4B). Since these regions are implicated in anti-codon binding, drTrpRS II and hTrpRS may share aspects of tRNA recognition. In addition, the loop preceding the KMSKS motif is three residues shorter in drTrpRS II and hTrpRS compared with the corresponding loop in bsTrpRS. This could have implications for the activation of adenylation (see below).
Incorporation of ATP into drTrpRS II Crystals-Although drTrpRS II was purified in the presence of 10 mM Mg-ATP and has a K D for Mg-ATP of ϳ10 M (8), Mg-ATP was found bound in the active center only if the crystals were soaked with 10 mM Mg-ATP 2 h prior to data collection. Higher concentrations of Mg-ATP were precluded in the soaking experiments by crystallization of ATP in the polyethylene glycol/phosphate buffer that stabilizes the crystals. No evidence was found for the binding of the nonhydrolyzable ATP analog ADPPNP under similar conditions. A difference electron density map (F o Ϫ F o ) calculated with diffraction data from the ATP-soaked crystals and data from crystals grown in the absence of additional ATP shows electron density for the bound nucleotide only in one of the three drTrpRS II subunits contained within the asymmetric unit (Fig. 5). In the most closed B subunit of the asymmetric dimer, the F o Ϫ F c electron density map derived from the refined structure also reveals clear density for the three phosphates, magnesium, and the ribose unit at 2.5 but only weak density for the adenine base at this contour level (Fig. 6). Thus, although ATP is present in a substantial fraction of the B subunits, it is either not completely occupied or partially disordered in the binding site. Accordingly, it is difficult to unambiguously differentiate the position of magnesium versus the triphosphate moiety based on the experimental electron density alone. However, the best fit of the nucleotide produces a conformation that is nearly identical to that of Mg-ATP in the active center of bsTrpRS (Fig. 6). The removal of magnesium from the model generates positive electron density peaks in Fourier difference maps surrounding the triphosphate group.
The Closed Conformer of drTrpRS II Binds ATP, whereas the Open Conformer Binds Trp-ATP makes similar contacts with the KMSKS and HLGH motifs of drTrpRS II as observed in the open complex of bsTrpRS with 1 mM ATP (Fig. 6, A and B) (12).
where F o and F c are the observed and the calculated structure factors, respectively, for 95% of the reflections uesd in the refinement. R free was calculated as for R work but on 5% of reflections excluded before refinement.
The carbonyl oxygen of Met 216 is aligned to hydrogen-bond to the amino group of the ATP adenine ring in both structures but is out of hydrogen bonding range in drTrpRS II, perhaps reflecting the weak density of the adenine base in this complex. Ser 217 and Ser 219 hydrogen-bond to the ␤and ␥-phosphates with their side-chain hydroxyl groups (as do the equivalent Ser 194 and Ser 196 residues in bsTrpRS). However, in the closed pre-TS conformation of bsTrpRS, the Ser 194 hydroxyl switches to hydrogen-bond with the Lys 192 side chain. Furthermore, the KMSKS loop moves closer to the nucleotide, allowing the main chain nitrogens of Lys 195 and Ser 196 region to coordinate the ␤and ␥-phosphates (Fig. 6C). In drTrpRS II, His 39 (of the HLGH motif) substitutes for bsTrpRS Asn 18 (of the analogous TIGN motif), but both residues interact with the respective ribose ring oxygen (Fig. 6B). Lys 215 and Lys 218 of the KMSKS loop, which bind the ␤and ␣-phosphate, respectively, in the bsTrpRS pre-TS, are disordered and project toward solvent in the drTrpRS II ATP complex (Fig. 6B). Thus, mobility of these lysine residues and the lack of hydrogen bonds between the KMSKS main chain and the ATP pyrophosphates indicate that the drTrpRS II active center has not yet progressed to a catalytically competent conformation in the absence of Trp. Fur-thermore, the disorder and/or less than complete occupancy of the ATP indicates that higher affinity binding may require a molecular conformation precluded by the crystal lattice. Nevertheless, the more open C subunit and crystallographically symmetric A subunit show no evidence of ATP binding.
Although the KMSKS loop does not completely engage ATP in the drTrpRS II structure, the RF domain makes interactions that closely resemble those in the bsTrpRS pre-TS. In particular, Arg 30 of ␤ 1 hydrogen-bonds to the ␣-phosphate of ATP (Fig.  6B), whereas the analogous bsTrpRS residue, Gln 9 , only contacts the ATP ␥-phosphate in the closed conformation (Fig. 6C). Arg 30 provides a key determinant for binding Trp derivatives in drTrpRS II by forming a salt bridge with Asp 68 (25). In general, all of the residues that contact Mg-ATP have similar conformations in both the subunits of the Trp-bound structure and the empty A subunit of the symmetric dimer. Thus, drTrpRS II does not require large conformational changes to bind Mg-ATP.
drTrpRS II binds Trp in a mode surprisingly different from how bsTrpRS recognizes substrates (Fig. 7). Most significantly, the indole ring in drTrpRS II tilts by ϳ45°, and the ␣-amino and ␣-carboxyl moieties exchange positions in the binding FIG. 1. A,  pocket (25). Several substitutions 2 in the active center of drTrpRS II mediate this change in binding orientation (25) (Fig. 7). In particular, Met 129 , which stacks against the Trp indole and ␣-amino group in most other TrpRSs, is replaced by Gln 154 , which instead hydrogen-bonds to the Trp carboxylate in the flipped orientation (25). Movement of highly conserved Gly 28 induced by three other substitutions (Asp 29 , Arg 47 , and Val 58 ) compensates for the tilted indole ring in drTrpRS II. Gln 64 , which participates in a hydrogen bond network with Arg 30 and Gln 154 , substitutes for a highly conserved Tyr 43 . Taken together, the unusual residues in the drTrpRS II binding pocket combine to expose the 4-and 5-position of the Trp indole within the active center channel, allowing drTrpRS II to accommodate modified indoles (25). Arg 30 , a residue conserved only by TrpRS IIs, interacts with both the ATP ␣-phosphate and the Trp amino acid group in the same conformation. This residue then couples the two binding sites and contributes to relative rigidity of TrpRS II.
Superposition of B and C molecules from the crystal structures of drTrpRS II bound to either ATP or Trp shows that only a ϳ1-Å movement of the TAB domian is required to bring the substrates together in a ternary complex (Fig. 8). However, for condensation to adenyl-Trp, Trp must flip its side chain to attack the ␣-phosphate of ATP. Thus, unlike bsTrpRS, drTrpRS II binds both ATP and Trp in similar conformations that require little further TAB domain motion to engage the substrates. Interestingly, the crystal structures indicate that in Residues that Limit Domain Motions in drTrpRS II-Limited domain motions in drTrpRS II are enforced by interactions supplied by other conserved residues. In bsTrpRS, Phe 26 and Tyr 65 serve as a bearing for domain movement; Phe 65 separates from Tyr 65 on TAB domain opening and instead contacts Leu 77 (15). In drTrpRS II, Arg 47 (which corresponds to Phe 26 of bsTrpRS), forms a salt bridge with Asp 29 in all three crystallographically unique subunits. Large relative motions of the RF and TAB domains will result in loss of this salt bridge without an obvious means for stabilizing the liberated charge partners. Furthermore, in bsTrpRS II a salt bridge between Glu 145 and Arg 182 reaches across the interdomain hinge to restrict the TAB domain from rotating further in the open state (12,15). The equivalent residues in drTrpRS II (Asp 171 and Arg 206 ) also interact in the more compact "open" state of drTrpRS II and thereby also restrict its conformation. Arg 206 in drTrpRS II projects closer to the RF domain than bsTrpRS Arg 182 because of an inserted proline residue at position 205. Unlike Arg 182 of bsTrpRS, Arg 206 also stacks against the adenine ring of ATP. Taken together, these TrpRS II-conserved residues (including Arg 30 mentioned above) provide interactions that limit TrpRS II domain motions relative to those of more typical bacterial TrpRSs.
Intersubunit Communication in drTrpRS II-Nearly all TrpRSs exhibit some form of subunit cooperativity on binding substrates (20); nevertheless, all known structures are highly symmetric and thus provide limited insight into how the active centers in adjacent subunits communicate. In contrast, asymmetry found of the BC drTrpRS II dimer results in Trp binding to only the B subunit. Because the crystallographically symmetric drTrpRS II dimer will not bind any ligands, Trp binding appears to require asymmetry between subunits. Half-site reactivity of drTrpRS II is also supported by kinetic and thermodynamic measurements. Steady-state kinetics of tRNA charging indicates that one Trp binds per TrpRS II dimer (n ϳ 0.5) (25). Furthermore, isothermal titration calorimetry of Trp binding to the drTrpRS II dimer gives a binding constant of 30 M with number of sites, n ϳ 0.5 (Fig. 9).
Differences in loops connecting secondary structural elements near the active site may impact binding of Trp, ATP, and tRNA. In the RF domain, drTrpRS II and hTrpRS have a three-residue longer loop connecting ␣ 6 and ␣ 7 (Fig. 2). This loop projects over the entry to the active center and participates in tRNA acceptor arm recognition in other TrpRSs. In drTrpRs II, the same region is essential to structural communication between Trp binding sites.
Overlaying the symmetric and nonsymmetric dimers of drTrpRS II (Fig. 10A) indicates that negative cooperativity may result from changes in active site residues on Trp binding that propagate to interactions at the dimer interface. Bound Trp in the C subunit interacts with residues on ␣ 8 , the so-called "substrate specificity helix" (19,29). Subtle structural changes draw nearby dimer interface helices ␣ 6 and ␣ 7 toward ␣ 8 and the bound ligand (Fig. 10). Asp 68 of ␣ 2 appears key to this transition. Upon Trp binding, Asp 68 switches the hydrogen bonding partner from Tyr 139 (on the loop connecting ␣ 6 and ␣ 7 ) to active site residue Arg 30 (Fig. 10B). As a result, the ␣ 7 -␣ 8 region moves toward the ␣ 8 specificity helix and active site in the Trp-bound subunit. To maintain tight interactions at the interface, ␣ 6 and ␣ 7 on the empty subunit shift toward the Trp-bound subunit and away from ␣ 8 . The increased gap between ␣6-␣7 and ␣2 in the empty subunit favors the buttressing hydrogen bond between Asp68 and Tyr 139 (Fig. 10C). Also in response to the ␣ 6 -␣ 7 shift, the side-chain hydroxyl of Ser 122 on the empty subunit swivels to hydrogen bond with the peptide carbonyl of Glu 141 across the dimer interface. Arg 125 and Glu 141 of the empty subunit hydrogen-bond with each other near the dimer interface. The position of this salt bridge appears to direct Arg 125 on the occupied subunit toward the Trp binding site. In all TrpRS II sequences, 2 positions corresponding to 125 and 141 contain a salt bridge pair; however, the acidic side chain can reside at either position. Such compensatory changes indicate that maintenance of an interacting charge pair at residues 125 and 141 has functional importance for TrpRS II.
Binding two Trp residues per dimer would generate strain by causing each subunit to effectively pull on the interface region from opposite directions, break both Asp 68 -Tyr 139 hydrogen bonds simultaneously, and direct the symmetric Arg 125 -Glu 141 salt bridges to the same region. This may be why Trp does not bind the A subunit, which is forced to be symmetric by crystallographic symmetry. Notably, standard bacterial TrpRSs, which do not show negative cooperativity with respect to Trp binding, have a shorter ␣ 6 -to-␣ 7 loop and do not conserve the same residues in these regions as TrpRS IIs.

FIG. 9. Isothermal titration calorimetry indicates that one Trp binds to one drTrpRS II dimer.
Heat released (kcal/mol) as a function of tryptophan concentration is least squares-fit to a one-site model.

DISCUSSION
TrpRS II Is Conformationally Rigid-Large amplitude domain motions are associated with the mechanism of substrate binding and adenyl-Trp formation by bsTrpRS. How applicable are these motions to drTrpRS II? ATP binds to two different conformations of bsTrpRS (open and closed), depending on its concentration and the pH (15). Increasing the ATP concentration and lowering the pH closes bsTrpRS to a conformation consistent with the pre-TS proposed for the TyrRS catalytic mechanism (15,17). This transition results from a 13°rotation of the TAB domain that moves the KMSKS region ϳ4 Å toward the RF domain and increases its interaction with ATP. In contrast, the free and ATP-bound structure of drTrpRS II adopts very similar conformations. In the drTrpRS II structure, the triphosphate moiety of ATP interacts with residues on the RF domain, despite interactions of the KMSKS loop more closely paralleling the open structure of bsTrpRS. Thus, the ATP complex of drTrpRS II shows features of both the bsTrpRS open and pre-TS structure. However, the less than full occupancy for ATP in drTrpRS II despite the high concentrations of nucleotide present may indicate that the conformation observed in the crystal is not optimum for binding ATP. The crystal lattice clearly influences the ability of the subunits to bind substrates, since subunits A and C show no evidence of ATP binding, and subunits A and B will not bind Trp. These subunits are not very different from each other in structure; thus, well ordered binding of ATP to the B subunit probably requires further conformational change either prevented by the lattice or gated by the addition of Trp (see below).
The greater rigidity of drTrpRS II probably stems from differences in sequence conserved by TrpRS IIs 2 rather than effects of crystal packing. As examples, 1) drTrpRS II Arg 30 mediates coupling between sites for ATP and Trp in a manner not replicated by equivalently positioned residues in bsTrpRS; 2) a restricting salt bridge formed between Arg 47 and Asp 29 replaces the pivot point for domain motion in bsTrpRS generated from Phe 26 and Tyr 65 ; and 3) Asp 171 and Arg 206 interact in the drTrpRS II open form much like their analogs Glu 145 and Arg 182 in bsTrpRS, despite the drTrpRS II open state having a more compact structure than that of bsTrpRS. In this latter case, the Asp 171 to Arg 206 salt bridge forms in drTrpRS II because the hinge region containing Arg 206 has an inserted proline residue that places Arg 206 closer to the RF domain than its counterpart in bsTrpRS. Thus, key regions that stabilize domain orientations differ in the two types of TrpRSs. However, one mobile region that shows high sequence conservation between drTrpRS II and bsTrpRS is the KMSKS loop, and thus the mechanism by which this region stages the adenylation reaction is also likely to be conserved.
drTrpRS II Is Structurally Closer to hTrpRS than to bsTrpRS-Although the RF domains of drTrpRS II, bsTrpRS, and hTrpRS are topologically quite similar, the TAB domain of drTrpRS II has much higher similarity to the hTrpRS TAB domain than to that of bsTrpRS (Figs. 4, A and B). Thus, the mechanism of tRNA recognition by drTrpRS II may be more closely related to that of eukaryotic than to other bacterial TrpRSs. This is somewhat surprising, because prokaryotic and eukaryotic AARS are usually orthogonal to each other in that they will not aminoacylate the respective tRNA (20). Despite the structural similarity, there is little sequence similarity between the drTrpRS II and hTrpRS TAB domains (Fig. 2), and in fact, the tRNA Trp from these organisms are also quite different. In the regions proposed to recognize tRNA, there is considerable sequence homology between drTrpRS I and drTrpRS II, which is consistent with the ability of both synthetases to aminoacylate the same in vitro transcripted D. radiodurans tRNA Trp (25).
The Essential Role of the KMSKS Loop in Acyl-adenylation by TrpRSs-The current model for adenyl-Trp formation by bsTrpRS incorporates a high energy pretransition state corresponding to a nearly closed conformation for the TAB domain (12,15). This places the KMSKS loop in an unfavorable conformation that destabilizes the ground state (12,15). At high ATP concentrations, the pre-TS of both subunits gains a number of protein-ligand interactions that energetically compensate for the anti-cooperative binding of the second ATP molecule. Under normal circumstances, with only one ATP molecule bound per dimer, the pre-TS forms only after binding both ATP FIG. 10. Intersubunit communication in drTrpRS II. A, stereoview of the superposition between the crystallographically symmetric drTrpRS II dimer interface (subunits colored in light blue and orange) and the asymmetric dimer (B subunit colored in blue; C subunit colored in yellow). Trp binds only to the C subunit and shifts the dimer interface region of both subunits toward the occupied site. B and C, side chain movements couple to main-chain shifts to propagate structural changes between active sites of the dimer. In comparison with the empty symmetric dimer (C), upon binding Trp (B), Asp 68 switches its hydrogenbonding partner from Tyr 139 to Arg 30 , Ser 122 (on subunit B) swivels to hydrogen-bond with the main chain of subunit C, and a salt bridge between Arg 125 and Glu 141 forms within the empty subunit. and Trp in the same subunit.
All three conformations of drTrpRs II have domain orientations that resemble the pre-TS rather than the open form of bsTrpRS; although interactions between the KMSKS loop and ATP more closely resemble those seen in the open form of bsTrpRS (12). In particular, the main-chain amide nitrogens and lysine side chains of the KMSMS loop have not yet engaged the nucleotide phosphates. Furthermore, the adenine base is disordered or not fully occupied, probably because Lys 215 has yet to stack against it, and the Met 216 carbonyl is too far to hydrogen-bond with the adenine amino group.
Unlike bsTrpRS, the ATP complex of drTrpRS II differs little in conformation from the empty structure, and only one subunit of the dimer binds nucleotide. The minimal conformational changes induced upon ATP binding in drTrpRS II correlate with a greater binding affinity of drTrpRS for nucleotide (K D ϳ10 M) (8), compared with bsTrpRS (K D ϭ 100 M) (12). In bsTrpRS, Trp binding induces a coordinated rotation of Tyr 125 , Gln 107 , and Gln 9 , which narrow the indole pocket and allow Tyr 125 to swing down and interact with the Trp ␣-amino group. Thus, Tyr 125 was proposed to serve as a gate, in that it provides access to Trp in the open conformation but closes down after the substrate has bound (12). Tyr 125 is conserved in all class Ib and class Ic synthetases, but its equivalent in drTrpRS II (Tyr 150 ) does not change conformation upon binding either ATP or Trp. Instead, Gln 154 (conserved only in eukaryotic and bacterial TrpRS IIs) binds the Trp carboxyl group. ATP binding probably gates conformational switching that closes the molecule in a manner independent from motion of Tyr 150 . Our inability to observe a more closed structure when the crystals were soaked with Trp and Mg-ATP suggests that either the product readily forms and hydrolyzes in the crystal or that the crystal lattice prevents complete closure to the ternary complex.
Nevertheless, drTrpRS II may still visit a higher energy conformation upon binding the second substrate and progressing toward the TS. However, the pre-TS of drTrpRS II probably will not differ greatly in domain juxtaposition from the ATP complex. Thus, the key conformational changes that poise the system for acyl-adenylation probably involve small movements that restructure the KMSKS loop. As has been shown by extensive mutant and kinetic studies in TyrRS, engagement of this mobile loop destabilizes the ground state and thereby requires expenditure of binding energy from the second substrate (17). Our structures indicate that a large amplitude motion of the TAB domain need not be coupled to these catalytically important loop rearrangements.
Subunit Communication in Type I AARSs-The asymmetric structure of the drTrpRS II dimer suggests how negative cooperativity of substrate binding can manifest in class Ic AARSs. The ␣ 6 -to-␣ 7 loop, which supplies the key interactions that link the respective active centers to the dimer interface is unique to eukaryotic and bacterial TrpRS IIs (Fig. 2), perhaps explaining why only this class of TrpRSs show such properties with respect to Trp binding (20). Although the salt bridges at positions 125 and 141 are well conserved by TrpRS IIs, Tyr 139 , which interacts with Asp 68 , is not. Thus, despite the switch of the Asp 68 hydrogen-bonding partner on binding Trp, other more subtle structural effects may also contribute to negative cooperativity. In general, substrate binding causes the helical cluster of ␣ 4 , ␣ 5 , ␣ 6 , and ␣ 7 on both sides of the dimer to shift toward the occupied site. Disruption of packing interactions at the dimer interface upon binding two Trps simultaneously may be enough to disfavor binding at the second site. B. stearother-mophilus TyrRS also shows half-site reactivity with respect to Tyr binding (17). TyrRS contains an analogous ␣ 6 -␣ 7 region that may convey effects of substrate binding across the dimer interface by a similar mechanism. Since the TrpRS II dimer is expected to recognize and aminoacylate only one tRNA per turnover (1), there was presumably little evolutionary pressure to select against negative cooperativity with respect to Trp binding.
TrpRS II as a Drug Design Target-Aminoacyl-tRNA synthetases have been attractive targets for antimicrobial drugs (30). In fact, drugs such indolmycin and TAK-083 effectively inhibit bacterial TrpRSs (31). However, bacteria that contain an auxiliary TrpRS II, such as Streptomyces coelicolor, are resistant to indolmycin (32). Furthermore, some human pathogens, such as Streptococcus pyogenes, have only one TrpRS that is more homologous to drTrpRS II (50% sequence identity) than typical bacterial TrpRSs. Interestingly, residue substitutions that render TrpRS II more like TrpRS I sensitize S. coelicolor TrpRS II to indolmycin (32). Thus, the crystal structures of drTrpRS II provide a structural basis for the development of potentially useful antibiotics against TrpRS IIs.