Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based*

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg2+ ion in a manner that allows it to assist in transition state stabilization. The Mg2+ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg2+·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg2+ coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg2+. Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS.

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (K m ϳ 2 M), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ϳ1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg 2؉ ion in a manner that allows it to assist in transition state stabilization. The Mg 2؉ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg 2؉ ⅐ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg 2؉ coordination in the human cytosolic (H c )TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in H c TrpRS with a yetto-be determined role for Mg 2؉ . Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to H c TrpRS.
The accumulation of resistance in pathogenic organisms over time and with prolonged drug use necessitates the continued development of new anti-infective therapeutics. Such developments can include modifications to current drugs that are active against exploited targets while counteracting current resistance mechanisms or novel compounds targeted against underexploited targets. One group of enzyme targets that has been validated but remains underexploited is the class of aminoacyl-tRNA synthetases (aaRSs). 2 Aminoacyl-tRNA synthetases maintain the fidelity of the genetic code by ensuring the charging of tRNA with its cognate amino acid via the following two-step reaction.

REACTION 2
All aaRS enzymes bind ATP and activate a specific amino acid by catalyzing the formation of an aminoacyl 5Ј-adenylate (aa-AMP) during the first step. This is followed by transfer of the activated amino acid to the 3Ј-end of the correct tRNA. Structural and mechanistic differences among the different aaRS enzymes as well as orthologs of individual synthetases make it possible to selectively modulate the activity of specific synthetases, e.g. prokaryotic over eukaryotic TrpRS (1). This makes the aaRS enzymes attractive targets for novel anti-infective therapeutics.
Any compounds intended for clinical use must be much less inhibitory against the eukaryotic orthologs of its intended target. Naturally occurring aminoacyl-tRNA synthetase inhibitors include indolmycin (TrpRS), granaticin (LeuRS), mupirocin (IleRS), and ochratoxin A (PheRS) (1)(2)(3)(4). Of these, mupirocin displays the required selectivity for prokaryotic over eukaryotic IleRS and has been developed for the treatment of infections in humans (5).
Indolmycin produced by Streptomyces griseus displays selective inhibition for prokaryotic TrpRS (9 nM; Escherichia coli) over eukaryotic TrpRS (4 mM; Bos taurus) (6). Problems with off-target effects on tryptophan metabolism have prevented its clinical use (7). However, if we could understand the molecular basis for the observed inhibition and selectivity we could exploit this information for the rational design of antibiotics targeted against TrpRS on pathogens.
Structurally, tryptophan and indolmycin are quite similar with a heterocyclic indole moiety at the root of each ligand (Fig.  1). Indolmycin differs from tryptophan in the following ways. (i) The carbon that is functionally equivalent to C␤ is substituted with a methyl group. (ii) The carbonyl carbon is part of an oxazolinone ring. (iii) The hydroxyl and amine groups of tryptophan are replaced by the nitrogen and oxygen atoms of the oxazolinone ring, respectively. (iv) The -NH-CH3 moiety attached to the oxazolinone ring does not have functionally equivalent atoms in tryptophan.
BsTrpRS is one of the most extensively characterized TrpRS enzymes. Mechanistically a Mg 2ϩ ion is linked to what appears to be a dissociative transition state for tryptophan activation (8 -10). During catalysis, the Mg 2ϩ ion helps compensate for the increased negative charge that develops on the PP i leaving group, resulting from breaking the ␣P-O-␤P bond. The Mg 2ϩ ion must move to be catalytically competent, but no proteinmetal interactions have been observed in any of the BsTrpRS crystal structures determined. Instead, a remote allosteric location, the D1 switch, must undergo significant conformational change to promote the Mg 2ϩ ion to a catalytically competent position. The metal moves closer to the PP i leaving group, whose charge is further stabilized in the transition state by the KMSKS loop. ATP binding is required for the conformational switching between the open and closed states that allows for catalysis. ATP-dependent induced fit closing of the active site brings ATP ϳ4 Å closer to tryptophan in a predominantly translational movement mediated by relative movement of the catalytic and anticodon-binding domains.
In the absence of ATP, tryptophan binding is promoted by hydrophobic van der Waals interactions,interactions with Phe 5 , and a hydrogen bond between the indole nitrogen and Asp 132 of the specificity helix. When both substrates bind, the tryptophan substrate undergoes a rotational movement that brings the indole ring deeper into the binding pocket and results in more stabilizing interactions between tryptophan and active site residues. This change is facilitated by the inward movement of the specificity helix that is not observed when only tryptophan is bound.
ATP-dependent induced fit rearrangement of the active site facilitates proper ATP positioning in BsTrpRS, and molecular dynamics simulations demonstrate that tryptophan is required to achieve the requisite movement of the ␣P in H c TrpRS (11,12). Even a modest substitution of tryptophanamide in place of tryptophan prevents the repositioning of ATP. These findings support the idea that H c TrpRS is intrinsically better at discriminating between tryptophan and its structural analogs than is BsTrpRS.
H c TrpRS uses different structural elements for substrate recognition than its prokaryotic orthologs (13). Such elements include an extended N terminus with a ␤1-␤2 hairpin structure shown to have a role in ATP binding as well as the amino acid activation reaction in H c TrpRS (14). In contrast to BsTrpRS, it is tryptophan binding that leads to induced fit rearrangement of the active site in H c TrpRS. There are a greater number of binding determinants for tryptophan recognition as eight direct and water-mediated hydrogen bonds with polar side chains stabilize tryptophan in the active site. It has been proposed that amino acid activation proceeds via an associative transition state in H c TrpRS with an unclear role of Mg 2ϩ in the catalytic transition state (11). However, comparison of the pre-transition (Protein Data Bank code 2QUI) and product states (Protein Data Bank code 2QUJ) shows that, as with BsTrpRS, the ␣P of ATP must move 5.3 Å to be in a position for nucleophilic attack by tryptophan.
Despite mechanistic and structural differences, BsTrpRS and H c TrpRS have comparable tryptophan binding affinities. However, these inherent differences between prokaryotic and eukaryotic TrpRS enzymes promote the binding of indolmycin to prokaryotic TrpRSs ϳ1500-fold while protecting eukaryotic TrpRSs from such inhibition by a comparable amount. Determining the structure of BsTrpRS bound by Mg 2ϩ ⅐ATP and indolmycin allowed us to probe the structural basis for indolmycin inhibition and selectivity. Specifically, we examined this structure along with the catalytically relevant structures of BsTrpRS and H c TrpRS deposited in the Protein Data Bank to answer the following questions. 1) What are the structural consequences of binding indolmycin? 2) Why is indolmycin a tight inhibitor of prokaryotic TrpRS? 3) Why is indolmycin not an inhibitor of eukaryotic cytosolic TrpRSs?

Experimental Procedures
Construction of pet28-His-BsTrpRS Vector-The full-length BsTrpRS sequence was PCR-amplified from a pet11 construct made previously in the laboratory. PCR primers contained restriction sites for BamHI and HindIII. The resultant PCR product was digested with BamHI and HindIII. A threeway ligation among the PCR product (BamHI/HindIII), double-stranded oligo encoding for the TEV site (NdeI/BamHI), and pet28b (NdeI/HindIII) yielded an expression vector for His-TEV-BsTrpRS.
Expression and Purification of His-BsTrpRS-BsTrpRS was expressed by autoinduction with BL21(DE3)pLysS cells at 37°C (15). The cells were pelleted at 4500 rpm for 30 min, resuspended in lysis buffer, and frozen at Ϫ20°C. Upon thawing, cells were sonicated and centrifuged (16,000 rpm, 4°C, 1 h). His-BsTrpRS was captured from the lysate on nickel-nitrilotriacetic acid resin and eluted with 0.3 M imidazole. Purified protein was cleaved overnight with TEV while dialyzing out the imidazole. The cleaved protein mixture was passed back over a nickel-nitrilotriacetic acid column to capture both uncleaved protein and His-TEV protease.
Active Site Titration-Active sites were titrated by following the loss of ATP to determine the fraction of molecules compe- tent for catalysis (16,17). The reaction was performed at 37°C in a final reaction mixture containing 5000 cpm/l [␥-32 P]ATP, 10 M ATP, 0.5 mM tryptophan, 5 mM MgCl 2 , and 0.05 unit/l inorganic pyrophosphatase. The reaction was initiated with enzyme at a final concentration of 3 M. At various time points between 10 s and 30 min, 3 l of the reaction were added to 6 l of quench buffer (sodium acetate, pH 5.3, 1% SDS) and placed on ice. Three microliters of each quenched reaction were spotted onto a cellulose-PEI TLC plate and run in 0.75 M KH 2 PO4, pH 3.5 with 4 M urea to separate 32 P i and [␥-32 P]ATP. Plates were developed using a Typhoon imager and analyzed with ImageJ (18) and JMP (19).
Michaelis-Menten Kinetics-The incorporation of [ 32 P]PP i into ATP was tracked either by TLC or filter binding after purification on charcoal (17). Reactions contained [ 32 P]PP i (5000 cpm/l for TLC and 400 cpm/l for the filter assay), 0.1 M Tris, pH 8.0, 70 mM ␤-mercaptoethanol, 5 mM MgCl 2 , 10 mM KF, 2 mM PP i , 2 mM ATP, and tryptophan ranging from 0.3 to 100 M. Reactions were initiated with enzyme at a final concentration of 30 nM.
Indolmycin Inhibition Assays-Inhibition assays were performed as described above; Michaelis-Menten experiments for tryptophan were performed in the presence of stoichiometric amounts of indolmycin (a gift from Pfizer, ca. 1994) to enzyme. Indolmycin to enzyme ratios of 1:5, 1:1, and 5:1 were used, and results were fitted to a competitive inhibition model (1) using JMP (19). Non-linear regression to Equation 1 allowed for determination of K m for tryptophan and K i for indolmycin.
Rate ϭ ͓Tryptophan͔ ϫ k cat Differential Scanning Fluorimetry (Thermofluor)-The effects of ATP, tryptophanamide (LTN), and indolmycin on the thermal stability of BsTrpRS were assessed by thermofluor. We showed separately 3 that differential scanning fluorimetry detects a conversion of TrpRS into a molten globule form that fully denatures only at higher temperature. The following saturating ligand concentrations were used to ensure a predominance of conformations corresponding to those observed in crystal structures: 5 mM ATP, 5 mM MgCl 2 , 10 mM LTN, and 600 M indolmycin. All reactions contained 8 M BsTrpRS, 50 mM NaCl, 5 mM ␤-mercaptoethanol, 50 mM Hepes, pH 7.5, and 0.15% SYPRO Orange in a final volume of 20 l. Fluorescence intensities were determined using an Applied Biosystems 7900HTFast Real Time PCR instrument, and data were analyzed with MATLAB (Mathworks) with routines developed by Visinets, Inc. The software was built as a pipeline of several m-files connected to provide full analysis of the data, including thermodynamic characterization and presentation of statistics. Fluorescence at each data point along a melting curve is assumed to be the sum of contributions from two states with probabilities p 1 and p 2 established by thermodynamic equilibrium between the two states.
where a 1 and a 2 are adjustable parameters representing intercepts, b 1 and b 2 are the slopes of the linear dependences of the initial and final states, and T is the Kelvin temperature. The pipeline consists of the following three parts.
Part A is reading the data from high throughput, 384-well, real time PCR files and transforming them into a matrix consisting of four columns: (i) number of the well from which temperature-dependent readings were taken, (ii) an index representing the protein variant, and finally the data, (iii) temperature and (iv) fluorescence readings.
Part B is fitting the thermofluor data to a thermodynamic model (Equations 3 and 4).
where ⌬G is the Gibbs energy difference between the two states and e Ϫ⌬G(T)/RT is the Boltzmann factor that determines the state probabilities p 1 and p 2 .
where ⌬H and ⌬S are the enthalpy and entropy changes between the states, c p is the heat capacity at temperature T, and ⌬c p is the heat capacity change between the two states at the melting temperature T m . Part C is independent determination of T m assuming that the state probabilities p 1 and p 2 can be estimated from distances between the intersection of the melting curve with vertical lines connecting the extrapolated linear final and initial slopes. Data initially worked up using both methods B and C agreed closely, and the analysis reported here follows C.
Crystallization, Data Collection, and Structure Determination-Crystals of seleno-Met-substituted BsTrpRS in complex with ATP, Mg 2ϩ , and indolmycin were grown by vapor diffusion against a reservoir of 1.4 M potassium citrate and 0.1 M Hepes, pH 7.4. Crystals were cryoprotected in Fomblin-Y and passed in a nitrogen airstream before plunging into liquid nitrogen. Data were collected remotely at Southeast Regional Collaborative Access Team (Beamline ID-22) using inverse beam geometry at 0.979 Å to obtain experimental phases from the Bijvoet differences and processed with XDS (20). PHENIX (21) and Coot (22) were used for phase determination, to interpret the map, and to iteratively refine the final structure (Protein Data Bank code 5DK4).

Results
BsTrpRS Binds Indolmycin ϳ1500ϫ More Tightly than Tryptophan-Indolmycin is a competitive inhibitor of Bacillus stearothermophilus and other bacterial TrpRS enzymes that competes with tryptophan for binding to the active site of the enzyme. By conducting Michaelis-Menten experiments at increasing tryptophan concentrations in the presence of different indolmycin concentrations and fitting all 64 data points simultaneously to Equation 1, we were able to determine K m,tryptophan (3 M) and K i,indolmycin (2 nM) ( Table 1). As these experiments were carried out under exchange conditions (23), we determined the standard free energy, ⌬G 0 ϭ ϪRTlnK, at 310 K for tryptophan and indolmycin binding to be 7.8 and 12.3 kcal/mol, respectively. This translates to a free energy difference of 4.5 kcal/mol between the affinities of the catalytic and inhibited complexes for the indole-containing ligand. To determine what factors account for the observed difference in binding free energy, we determined the structure of the BsTrpRS⅐ATP⅐indolmycin ternary complex and then conducted differential scanning fluorimetric experiments in the presence of various ligands.
A previously unpublished structure of BsTrpRS bound to Mg 2ϩ ⅐ATP and indolmycin was never deposited (27). Nevertheless, that structure was the first example of a series of subsequently solved structures that have been described as "pretransition state" (PreTS) structures (Protein Data Bank codes 1MAU and 1M83 (12)). In these structures, the initial ATP binding site in the small domain composed of the N-terminal ␣-helix and the anticodon-binding domain closes on the remainder of the Rossmann fold, bringing the nucleotide ␣-phosphate from 6.7 Å away to within van der Waals contact distance of the tryptophan carboxyl oxygen (12).
The new structure presented here is at higher resolution (1.9 versus 2.4 Å), and the experimental phases greatly enhanced the quality of electron density maps (Table 2 and Fig. 2). Details of the new structure, such as the orientation of the ribose and the metal position, are quite similar to those observed in deposited PreTS structures 1MAU and 1M83. Detailed differences that appear functionally relevant are discussed below.
Indolmycin Induces New Contacts with Active Site Side Chains-Indolmycin makes contacts with the side chains of His 43 , Asp 132 , and Gln 147 as well as two water molecules (Fig.  3A). The interaction between O␦2 of Asp 132 in the specificity helix and the nitrogen atom of the indole ring is observed when tryptophan (3.1 Å), tryptophanamide (3.0 Å), or indolmycin (2.9 Å) is bound. The addition of the oxazolinone group to the ligand allows for stabilizing interactions with His 43 and Gln 147 with functionalities on either side of the ring, which have the effect of fixing the rotation of the ring (Fig. 3B). N␦1 of His 43 can donate and/or accept a hydrogen bond from N2 (methylamino group) of indolmycin. In addition to these hydrogen bonds, His 43 can form a salt bridge with O␦2 of Asp 132 (2.8 Å). The amide group of Gln 147 forms two hydrogen bonds, one with the carbonyl oxygen of the oxazolinone ring (3.0 Å) and another with O␦2 of Asp 146 (3.0 Å). O␦1 of Asp 146 makes a highly conserved hydrogen bond with the 2Ј-OH group of ATP (2.7 Å). These side chain interactions in the conserved GEDQ motif link the indolmycin and ATP binding sites while reinforcing the linkage between opposite sides of the indole-binding pocket.
Structurally, indolmycin binding also prevents the Tyr 125 rotamer switch that occurs upon the enzyme going from its open to closed conformation (Fig. 4, A and B

Steady-state kinetic analysis of indolmycin inhibition
Non-linear regression analysis of 64 data points from steady-state kinetics experiments confirms indolmycin as a tight binding competitive inhibitor of BsTrpRS. PP i exchange assays performed in the presence of saturating Mg 2ϩ ⅐ATP with varying concentrations of tryptophan and indolmycin show that BsTrpRS binds indolmycin ϳ1450 times more tightly than tryptophan. The difference in free energy between the catalytic and inhibited complexes is ϳ4.5 kcal/mol.  In the pre-transition state, the N atom of Lys 111 forms a salt bridge with the O␥1 atom of ATP (2.9 Å), which also forms a strong electrostatic interaction with the catalytic Mg 2ϩ ion (2.4 Å). This interaction presumably is important for stabilizing the developing charge on the PP i leaving group released after tryptophan activation. Additionally, in the pre-transition state, Lys 111 is in position to act as a hydrogen bond donor to the one water molecule coordinated to the Mg 2ϩ ion (Fig. 5). The subtle opening of this loop in the inhibited state weakens the salt bridge between the Lys 111 N atom and the O␥1 atom of ATP (3.4 Å) from that observed in the preTS. Now the closest interactions are with three water molecules (2.6, 2.8, and 2.8 Å), none of which are coordinated directly to the catalytic Mg 2ϩ ion so they are not shown in Fig. 5.
Replacement of tryptophan(amide) with indolmycin in the active site alters the coordination and placement of the Mg 2ϩ ion used during the activation step of the aminoacylation reaction. Presumably because its orientation is fixed by the hydrogen bonding network described above, the oxazolinone forms hydrogen bonds with two water molecules. Introduction of these two water molecules is associated with the movement of the Mg 2ϩ ion into a hexavalent coordination that closely resembles stable configurations generated in quantum mechanical simulations of Mg 2ϩ ⅐ATP. 4 As is also true in the PreTS structure, the Mg 2ϩ ion in the inhibited BsTrpRS structure coordinates with a non-bridging oxygen from each phosphate group and three water molecules (Figs. 3B and 4B), two of which are further stabilized by the presence of indolmycin. In addition to the three electrostatic interactions with ATP, only one water molecule was seen to 4 S. Liu, unpublished data.  coordinate with the Mg 2ϩ ion in the PreTS structure (Protein Data Bank code 1MAU) (Figs. 4A and 5). The side chain residues that accept and donate hydrogen bonds to this water molecule differ between these two states (Fig. 5). Due to the different Gln 107 rotamer and slight opening of the mobile loop around Lys 111 , these two residues no longer interact with this water molecule in the inhibited state. The Mg 2ϩ ion is closer and more central to the triphosphate moiety in the inhibited structure than in the PreTS structure Ligand-induced Stability Changes Imply Cooperative Sources of High Indolmycin Affinity-The nature of the ligands within the active site has a significant effect on the conformation and thermal stability of an enzyme. For small perturbations, the fractional change in melting temperature (⌬T m /T m ) induced by ligand binding is proportional to the free energy change in stability, the proportionality constant being the enthalpy change (⌬H) (28). We use this implicit relationship to assess the stabilizing or destabilizing effects of various ligands on the BsTrpRS enzyme. Binding of ATP, tryptophan, or tryptophanamide stabilizes the thermal transition of molten globule formation by 3, 7, and 7%, respectively (Table 3). Indolmycin enhances the thermal stability of BsTrpRS by 20%, increasing T m by 13.5°C. The enhanced affinity for indolmycin over tryptophan results in a shift by 8°C to higher temperature in the thermal transition due to molten globule formation in the presence of indolmycin compared with tryptophan.
The linkage between protein stability and ligand binding (29 -31) implies that we can attribute differences in stability changes to binding affinity. Two of the stabilizing interactions formed between indolmycin and Mg 2ϩ -coordinated water molecules are associated with a change in the metal position relative to the ATP phosphate oxygen atoms. A key implication of the structural observations in Figs. 3A and 4C is that binding of indolmycin to BsTrpRS should be potentiated by the presence of Mg 2ϩ ⅐ATP. A, compared with the apo form (pink; Protein Data Bank code 1D2R), the fully occupied PreTS structure (black; Protein Data Bank code 1MAU) assumes a closed conformation. The C␣ of Tyr 125 is shifted inward by 2.4 Å, and the side chain is flipped ϳ45°(measured from OH-Ca-OH). A Mg 2ϩ ion (black sphere) forms electrostatic interactions with ATP and one water molecule (salmon sphere). B, binding of indolmycin and ATP causes similar shifts in the backbone (gray) as the enzyme adopts a closed conformation. However, due to the addition of the methylamino-substituted oxazolinone ring, this movement to the closed conformation is not accompanied by a rotamer change of Tyr 125 in the inhibited structure. C, consequently, Gln 107 is constrained and is rotated 106°around C␤ away from the specificity helix in the inhibited state compared with the pre-transition state. Finally, the Mg 2ϩ (green sphere) is shifted toward the ␣PO 4 and has hexavalent coordination to ATP and three water molecules (red spheres) as compared with the preTS structure. The presence/position of Mg 2ϩ in the active site is strictly dependent on ATP because the protein makes no contacts with the metal. As no direct ATP-indolmycin interactions are observed, we therefore expected that ATP would enhance the thermal stability of the BsTrpRS⅐indolmycin complex by a larger amount in the presence compared with the absence of Mg 2ϩ . Additionally, we did not expect Mg 2ϩ to contribute to thermal stability in the absence of ATP. As expected, the differential scanning fluorimetry measurements show that the BsTrpRS in complex with indolmycin and Mg 2ϩ ⅐ATP has a 27% increase in melting temperature compared with ligand-free enzyme with Mg 2ϩ ⅐ATP contributing an additional 5°C of thermal stability on top of the 13.5°C provided by indolmycin binding. By contrast, binding of indolmycin, indolmycin ϩ Mg 2ϩ , or indolmycin ϩ ATP all elicit far smaller changes of ϳ20% in thermal stability, demonstrating that both Mg 2ϩ and ATP are required to confer additional thermal stability to the BsTrpRS⅐indolmycin (IND) complex.
The conclusion that the metal is essential to the enhanced affinity of indolmycin to the pre-transition state complex can also be derived using the three-dimensional thermodynamic cycle of contributions to stability from ATP, the methylaminosubstituted oxazolinone ring (OXA), and the presence/absence of Mg 2ϩ (Fig. 6). Differences in binding and thermal stability between tryptophanamide and indolmycin were attributed to the methylamino-substituted oxazolinone ring as this is the major structural difference between these two ligands.
Stabilizing and destabilizing interactions are distinguished by positive and negative non-additivity, respectively. If thermal stability were unaffected by interactions between the ligands, then we expect the effects of binding multiple ligands, e.g. ATP and LTN, to be additive and the effects of binding one ligand not to be affected by the presence of a second ligand, thus giving Equations 5 and 6.
An interaction between the ligands would introduce a term, ⌬T m,int, to describe the non-additivity (32), giving Equation 7.
In the absence of Mg 2ϩ , there is no significant ATP-OXA interaction, and binding either IND or LTN reduces the effect of ATP on T m by ϳ1.5°C. The ATP-LTN and ATP-IND interactions are both destabilizing; i.e. the doubly liganded complexes melt at lower temperatures. In contrast, addition of Mg 2ϩ stabilizes the interactions of ATP with LTN and IND to varying degrees. In the case of LTN-ATP, addition of Mg 2ϩ compensates for the destabilizing ATP-LTN interaction such that the interaction is no longer significant. The metal compensates for the Ϫ1.3°C destabilizing ATP-IND interaction and allows for an additional stabilizing interaction of 2.6°C. Thus, the ⌬T m (ATP-OXA) interaction is comparable with that of the ⌬T m (ATP-IND) interaction. The crystal structure suggests that the stabilizing effect of Mg 2ϩ on the interaction In the presence of Mg 2ϩ (bottom row), the interaction between ATP and LTN is insignificant, whereas the ATP-IND interaction raises the T m 4% higher than expected. As expected from the crystal structure, the Mg 2ϩ -dependent ATP-IND interaction is mediated through the oxazolinone moiety of indolmycin. LF, ligand-free.

TABLE 3
Thermofluor analysis of ligand-dependent stability between ATP and indolmycin is mediated through the oxazolinone ring, whose orientation is, in turn, stabilized by hydrogen bonds to His 43 and Gln 147 as discussed above.

Discussion
An array of crystal structures of both BsTrpRS and H c TrpRS provide snapshots of the enzymes along their catalytic paths and demonstrate the conformational changes that result from binding of various ligands (12,13,24). From these structures, it is evident that H c TrpRS uses a greater number of binding determinants for tryptophan recognition and that binding of tryptophan causes an induced fit rearrangement of the active site in H c TrpRS but not BsTrpRS. Here we discuss possible structural and mechanistic reasons for the tight binding of indolmycin to BsTrpRS and the inability of indolmycin to inhibit eukaryotic TrpRSs.
Why Is Indolmycin a High Affinity Inhibitor of Bacterial TrpRS?-There are no drastic global changes between structure 1MAU and the inhibited BsTrpRS structure (Protein Data Bank code 5DK4). We propose that subtle, mechanistically relevant differences in the active site metal configuration account for the ability of indolmycin to inhibit BsTrpRS as tightly as it does. We observe stronger Mg 2ϩ -ATP and weaker BsTrpRS-ATP interactions (Fig. 6) as well as altered Mg 2ϩ coordination and placement in the inhibited state (indolmycin ϩ Mg 2ϩ ⅐ATP; Figs. 3B and 4) compared with the pre-transition state (tryptophanamide ϩ Mg 2ϩ ⅐ATP) structure. We attribute these differences to the replacement of tryptophanamide with indolmycin that varies mainly at the methylamino-substituted oxazolinone ring of indolmycin. Interactions among His 43 , Gln 147 , and indolmycin restrict the oxazolinone ring orientation, thereby reducing the entropy of the ␣-carbon mimic in the inhibited complex compared with the pre-transition state complex. This unfavorable entropy change is compensated by the enthalpy from additional hydrogen bonds formed among the Mg 2ϩ -coordinated water molecules and the oxazolinone nitrogen and carbonyl oxygen atoms as well as the interaction with His 43 .
These hydrogen bonds stabilize the water molecules that are also tightly coordinated to the catalytic Mg 2ϩ ion. Functional groups of the ␣-carbon atoms of tryptophan and tryptophanamide can adopt alternative conformations that are similar in energy, none of which allow for completion of the Mg 2ϩ coordination sphere. We conclude from these observations that completion of that coordination sphere allows the metal to form significantly tighter interactions with all three phosphate oxygen atoms and hence that indolmycin stabilizes a ground state Mg 2ϩ ⅐ATP configuration, opposing the tendency of the PreTS state to promote the metal to a high energy state that assists in transition state stabilization.
Furthermore, the oxazolinone ring of indolmycin, stabilized by hydrogen bonds with His 43 and Gln 147 , prevents the rotamer switch of Tyr 125 in the specificity helix that is part of the structural transition from the open to the closed state. To avoid a steric clash with the constrained Tyr 125 residue, Gln 107 likewise does not switch rotamers in the presence of indolmycin. Gln 107 is part of a highly mobile loop that shows a subtle but significant opening in the inhibited state compared with the pre-transition state. This opening results in the weakening of ATP-BsTrpRS interactions, specifically those between Lys 111 and the ␥-phosphate group.
In the catalytically competent PreTS configuration, coordination by lysine residues of the phosphate oxygen atoms promotes the metal to an activated, less stable state with weaker interactions to the three phosphate oxygen atoms and prevents the Mg 2ϩ ion from assuming a lower energy position with stronger contacts to ATP. The positively charged N atom of Lys 111 competes with the Mg 2ϩ ion for stabilization of a negatively charged oxygen atom (O␥) of the ␥-phosphate group. In the PreTS state, the Mg 2ϩ -O␥ and Lys 111 -O␥ distances are 2.4 and 2.9 Å, respectively.
Substitution by indolmycin for tryptophanamide simultaneously weakens the Lys 111 -O␥ (3.4 Å) interaction and strengthens that between that oxygen atom and the Mg 2ϩ ion (2.2 Å). Additionally, the 0.4-Å shift in Mg 2ϩ placement along with the opening of the mobile loop around Lys 111 allows for tight, hexavalent Mg 2ϩ coordination accompanied by stronger, more nearly equivalent interactions between Mg 2ϩ and the three ATP phosphate groups.
Mutation of His 43 Results in Indolmycin Resistance-When both tryptophanamide and ATP are bound, the His 43 side chain switches from one rotamer to another in the transition from open to closed PreTS state and back again in the closed product state. This rotamer switch does not occur when the active site is bound to AMP, PP i , and tryptophan (33). In the PreTS (Mg 2ϩ ⅐ATP ϩ LTN) and inhibited (Mg 2ϩ ⅐ATP ϩ IND) states, N⑀2 of His 43 interacts with O␦2 of Asp 132 . In all other observed states, N⑀2 of His 43 forms an interaction with the carbonyl oxygen of Tyr 125 . His 43 also contributes to indolmycin binding via N␦1. This reorientation of His 43 appears to be correlated with the succession of ligands most similar to the putative catalytic reaction path, and it may thus also be functional.
Several groups have identified mutations that confer high level indolmycin resistance (34 -36). One of the mutant sites, His 43 , is of direct interest in the context of the present inhibited structure, which furnishes a semiquantitative explanation for the mutational effects at position 43. We have implicated His 43 in a hydrogen bond network that requires hydrogen bonds to both indole nitrogen atoms that stabilize the orientation of the plane of the oxazolinone ring of indolmycin. Fixing the orientation of the ring consequently allows formation of a full hexacoordinated environment for the catalytic Mg 2ϩ ion, which we have shown accounts for the additional stabilization of the indolmycin⅐Mg 2ϩ ⅐ATP complex.
Indolmycin inhibition of the B. stearothermophilus TrpRS H43N mutant is weaker by 3.5 kcal/mol than that of the native enzyme (37). Depending on the stabilization energy provided by these two hydrogen bonds and their coupling, we would predict that an H43N mutant would lose 2-4 kcal/mol binding energy compared with wild-type enzyme. All rotamers of an asparagine substitution at position 43 would result in the loss of two of the three hydrogen bonds observed in the network among indolmycin, His 43 , and Asp 132 . Similarly, the homologous H48Q mutation in Streptomyces coelicolor similarly appears incapable of forming both hydrogen bonds we observe for His 43 (36).

Modeling Reveals Why Indolmycin Is a Weak Inhibitor of Eukaryotic Cytosolic TrpRS Enzymes-
The selectivity ratio of indolmycin for cytosolic B. taurus (Bt)TrpRS versus BsTrpRS is 10 6 -fold in favor of BsTrpRS binding. This selectivity factor far surpasses those of most therapeutic drugs, including trimethoprim (selectivity ratio rat/Toxoplasma gondii dihydrofolate reductase, 49) and metoprolol (selectivity ratio ␤ 2 /␤ 1adrenergic receptor, 6.0), which treat toxoplasmosis and cardiovascular disease, respectively (38, 39). This dramatic selectivity arises by enhancing indolmycin binding recognition by BsTrpRS as we have just shown while reducing eukaryotic cytosolic TrpRS affinity by similar magnitudes. H c TrpRS shares 93% sequence identity with BtTrpRS and is therefore highly likely to have a comparably weak millimolar affinity for indolmycin. Interest in developing indolmycin as a lead compound for anti-infective therapy as well as the extensive kinetic and structural studies conducted on H c TrpRS led us to examine the repertoire of deposited H c TrpRS structures with the purpose of identifying potential means by which eukaryotic cytosolic TrpRS enzymes evade inhibition by indolmycin (13,14,40,41).
Whereas BsTrpRS uses an induced fit mechanism for ATP binding, H c TrpRS uses induced fit for tryptophan binding. Binding of tryptophan to BsTrpRS is stabilized by one hydrogen bond between the indole nitrogen of tryptophan and O␦2 of Asp 132 andinteractions with Phe 5 . Meanwhile, H c TrpRS makes seven direct and water-mediated contacts to its tryptophan substrate. The determinants for tryptophan binding to H c TrpRS include Glu 199 , which has a direct and water-mediated interaction with the ␣-amino group of tryptophan (Fig.  7A). Modeling of indolmycin into the amino acid binding site introduces steric clashes between indolmycin and Glu 199 (Fig.  7B). Furthermore, Glu 199 cannot adopt an alternative rotamer conformation without introducing additional clashes between Glu 199 and Thr 196 , Trp 203 , or Phe 280 . Besides clashing with Glu 199 , this indolmycin conformer would not form any of the hydrogen bonds observed when tryptophan is bound aside from the bifurcated hydrogen bond with the indole nitrogen, Tyr 159 , and Gln 194 . These interactions are preserved because indolmycin was modeled into the active site by overlaying its indole moiety with that of tryptophan (Protein Data Bank code 2QUH).
Indolmycin can be modeled into the active site of H c TrpRS by rotating the oxazolinone ring away from Glu 199 to an orientation perpendicular to the indole moiety (Fig. 7B). Although this indolmycin conformer does not clash with active site residues, it does disrupt the hydrogen bonding pattern used by the cytosolic enzyme to identify tryptophan as the bound substrate. The ␣-amino group of tryptophan, which is protonated at physiological pH, is recognized by Glu 199 , Gln 284 , and Gln 313 (Fig.  7A). Each of these residues acts a hydrogen bond acceptor, and the negatively charged carboxylate of Glu 199 forms additional electrostatic interactions with the amino group. According to our model, the side chain amide group of Gln 284 would act as a hydrogen bond donor for the cyclic oxygen atom in indolmycin that is in the equivalent position of the ␣-amino group (Fig. 7B). Although Glu 199 cannot form a salt bridge with indolmycin, it can instead share a bifurcated hydrogen bond from the meth-ylamino group nitrogen with Gln 313 . This nitrogen can accept a hydrogen bond from the hydroxyl group of Tyr 316 .
Finally, Lys 200 cannot form a salt bridge with indolmycin as it does with the tryptophan carboxylate. This electrostatic interaction is also missing when tryptophanamide is bound in place of tryptophan and appears to be critical for progression from the pre-transition state to transition state as this is the only interaction used for tryptophan substrate recognition and binding that cannot form when tryptophanamide occupies the active site. Indolmycin differs from tryptophan by a greater degree than does tryptophanamide. The inability of indolmycin to fully retain the tryptophan-H c TrpRS side chain interactions, including the salt bridge with Lys 200 , allows H c TrpRS to discriminate between tryptophan and the inhibitor. For these reasons, a stable H c TrpRS⅐ATP⅐indolmycin complex is ϳ1000 less likely to form than that of H c TrpRS⅐ATP⅐tryptophan. Contrastingly, BsTrpRS, which has a 10 3 -fold higher affinity for indolmycin over tryptophan, is ϳ1500 more likely to form a stable, inhibited complex than a catalytically competent tryptophanbound complex. Rigid body modeling shows that none of the interactions important for tryptophan substrate recognition/binding can form between active site residues and indolmycin (yellow sticks). Additionally, rotating the oxazolinone ring away from Glu 199 eliminates a prominent steric clash between the methylamino group of indolmycin and the side chain carboxylate group of Glu 199 . This alternate indolmycin conformation (pink sticks) allows for more hydrogen bonding interactions between indolmycin and active site residues, although the nature of these interactions is different from those observed upon tryptophan binding. Modeling suggests that these altered interactions allow H c TrpRS to reject indolmycin as a substrate.
In this work, we determined the structural basis for high affinity inhibition of BsTrpRS by indolmycin. The simultaneous binding of indolmycin and Mg 2ϩ ⅐ATP results in (i) movement of the Tyr 125 and Gln 107 side chains, (ii) opening of the mobile loop containing Lys 111 , (iii) displacement of the Mg 2ϩ ion by 0.4 Å, (iv) hexavalent metal coordination, (v) stronger, nearly equivalent electrostatic interactions of Mg 2ϩ with an oxygen from each phosphate group of ATP, and (vi) weaker coordination of phosphate group oxygen atoms by active site lysine residues. These changes are reinforced by the hydrogen bonding interactions of Gln 147 , His 43 , and Mg 2ϩ -coordinated water molecules with indolmycin. We propose that weaker coordination by lysine residues of the phosphate oxygen atoms and stronger Mg 2ϩ -ATP interactions induced by indolmycin binding allow the Mg 2ϩ ion to settle into a lower energy state, thereby significantly increasing affinity by preventing activation of the metal required for use in amino acid activation.
Author Contributions-C. W. C. and T. L. W. conceived the experimental plan based on previous work by Y. W. Y., T. L. W. carried out all experimental work, analyzed the data, and wrote the paper in consultation with C. W. C. and Y. W. Y.