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

Abstract

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 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+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) 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 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 HcTrpRS.

Keywords: aminoacyl-tRNA synthetase; enzyme catalysis; enzyme inhibitor; inhibition; inhibition by ground-state stabilization; inhibition mechanism; thermofluor; x-ray crystallography.

FIGURE 1.

Functional equivalences of tryptophan and indolmycin. Indolmycin differs from tryptophan in three key ways: 1) the incorporation of Cα constituents into an oxazolinone ring, 2) a methylamino group extending from the oxazolinone ring, and 3) replacement of a hydrogen on Cβ with a hydrophobic methyl group.

FIGURE 2.

BsTrpRS forms a ternary complex with indolmycin and Mg²⁺·ATP in a closed conformation. A, functionally dimeric BsTrpRS crystallizes with one monomer in the asymmetric unit. B, in addition to indolmycin and ATP, the active site contains a Mg²⁺ ion and three stable water molecules. The F_o − F_c omit map, derived by omitting indolmycin, ATP, Mg²⁺, and three water molecules from a final round of refinement, contoured to 4.0 σ is depicted in green.

FIGURE 3.

Hydrogen bonding and electrostatic interactions promote indolmycin binding and Mg²⁺ coordination within the active site. A, BsTrpRS residues His⁴³, Asp¹³², and Gln¹⁴⁷ make stabilizing contacts with IND via side chain atoms. The ATP and indolmycin binding subsites are linked via a hydrogen bond (2.7 Å) between Asp¹⁴⁶ and Gln¹⁴⁷ of the conserved GXDQ motif. B, Mg²⁺ forms tight electrostatic interactions (2.0–2.3 Å) with three water molecules (red spheres) and an oxygen atom from each phosphate group of ATP. Two of these water molecules are within hydrogen bonding distance (2.6 and 2.7 Å) of indolmycin.

FIGURE 4.

Comparison of ligand-free, preTS, and inhibited BsTrpRS structures. 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 Cα of Tyr¹²⁵ is shifted inward by 2.4 Å, and the side chain is flipped ∼45° (measured from OH-Ca-OH). A Mg²⁺ 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 Tyr¹²⁵ in the inhibited structure. C, consequently, Gln¹⁰⁷ is constrained and is rotated 106° around Cβ away from the specificity helix in the inhibited state compared with the pre-transition state. Finally, the Mg²⁺ (green sphere) is shifted toward the αPO₄ and has hexavalent coordination to ATP and three water molecules (red spheres) as compared with the preTS structure.

FIGURE 5.

Differential BsTrpRS side chain interactions with the water molecules electrostatically coordinated with Mg²⁺ in the PreTS and inhibited structures. Gln⁹, Gln¹⁰⁷, and Lys¹¹¹ are in position to accept (Gln¹⁰⁷) or donate (Gln⁹ and Lys¹¹¹) hydrogen bonds (black dashed lines) to the water molecule (salmon sphere) coordinated with Mg²⁺ (black sphere) in the PreTS (black sticks). As indolmycin binding leads to opening of the mobile loop containing Gln¹⁰⁷ and Lys¹¹¹, these residues are too far (red dashed lines) to form stabilizing interactions with the equivalent water molecule (red sphere) in the inhibited complex (gray sticks). The three water molecules (red spheres) electrostatically coordinated to Mg²⁺ (green sphere) in the inhibited complex are stabilized by interactions (cyan dashed lines) with side chain atoms of residues Gln⁹, Asp¹⁴⁶, Gln¹⁴⁷, and Lys¹⁹².

FIGURE 6.

Contributions of Mg²⁺, ATP, indolmycin, and tryptophanamide to the thermal stability of BsTrpRS. TrpRS enzymes require a Mg²⁺ ion for tryptophan activation. Although Mg²⁺ does not change the thermal stability of BsTrpRS on its own, its presence or absence significantly impacts the interaction of ATP with both LTN and IND. In the absence of Mg²⁺ (top row), both the ATP-LTN and ATP-IND interactions lower the fractional change in T_m by 2% from its expected value. In the presence of Mg²⁺ (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 Mg²⁺-dependent ATP-IND interaction is mediated through the oxazolinone moiety of indolmycin. LF, ligand-free.

FIGURE 7.

Steric hindrance and altered hydrogen bonding pattern allow H_cTrpRS to discriminate between tryptophan and indolmycin. A, the electrostatic and hydrogen bonding interactions that facilitate specific recognition and binding of Trp to H_cTrpRS (Protein Data Bank code 2QUH) are shown in yellow dashes. B, IND (yellow and pink sticks) from the BsTrpRS complex was modeled into the tryptophan binding site of H_cTrpRS (Protein Data Bank code 2QUH) so the indole moieties superimposed. This maintained the H-bonding interactions between the indole nitrogen and Tyr¹⁵⁹ and Gln¹⁹⁴. 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 Glu¹⁹⁹ eliminates a prominent steric clash between the methylamino group of indolmycin and the side chain carboxylate group of Glu¹⁹⁹. 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_cTrpRS to reject indolmycin as a substrate.