Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli

Abstract

The tRNA modification N6-threonylcarbamoyladenosine (t6A) is universally conserved in all organisms. In bacteria, the biosynthesis of t6A requires four proteins (TsaBCDE) that catalyze the formation of t6A via the unstable intermediate l-threonylcarbamoyl-adenylate (TC-AMP). While the formation and stability of this intermediate has been studied in detail, the mechanism of its transfer to A37 in tRNA is poorly understood. To investigate this step, the structure of the TsaBD heterodimer from Escherichia coli has been solved bound to a stable phosphonate isosteric mimic of TC-AMP. The phosphonate inhibits t6A synthesis in vitro with an IC50 value of 1.3 μM in the presence of millimolar ATP and L-threonine. The inhibitor binds to TsaBD by coordination to the active site Zn atom via an oxygen atom from both the phosphonate and the carboxylate moieties. The bound conformation of the inhibitor suggests that the catalysis exploits a putative oxyanion hole created by a conserved active site loop of TsaD and that the metal essentially serves as a binding scaffold for the intermediate. The phosphonate bound crystal structure should be useful for the rational design of potent, drug-like small molecule inhibitors as mechanistic probes or potentially novel antibiotics.

Figure 1.

Biosynthesis of t⁶A in bacteria. In most bacterial tRNAs, t⁶A is further cyclized to the hydantoin ct⁶A, which is hydrolytically unstable to commonly used isolation procedures.

Figure 2.

Chemical structures of TC-AMP and inhibitors prepared and analysed in this work.

Figure 3.

Synthesis of phosphonate inhibitor BK951. See Materials and Methods for experimental details.

Figure 4.

Kinetics of E. coli t⁶A formation in the full reaction and isolated second step with purified TC-AMP as substrate. (A) Full reaction mixture with L-threonine as variable substrate fit to MM kinetics (Equation 1). (B) Full reaction mixture with ATP as variable substrate ≤1 mM. (C) Full reaction with ATP as variable substrate showing substrate inhibition at high concentrations (fit to Equation 3) (D) Full reaction with tRNA^Lys_UUU as the variable substrate fit to Morrison Equation (2). (E) Isolated second step of reaction with TC-AMP as variable substrate and 1 mM ATP fit to Morrison equation (Equation 2). (F) Second step of the reaction with TC-AMP (10 μM) as substrate and variable ATP displaying substrate inhibition as in (C). (G) Second step of the reaction with constant TCAMP (10 μM) and ATP as the variable substrate (≤1 mM) fit to MM Equation (1).

Figure 5.

Characterization of BK951 inhibition of t⁶A formation. (A) IC₅₀ determination for BK951 with full reaction mixture. (B) IC₅₀ determination for BK951 with 10 μM TC-AMP, 0.5 μM EcTsaBDE and 1 mM ATP. (C) K_d measurement of TsaB binding to fluorescently labelled TsaD using Microscale Thermophoresis (MST). (D) K_d measurement of BK951 binding to labelled TsaBD heterodimer using MST. (E) K_d measurement of TsaE to labelled TsaBD in the presence of 100 μM ATP. For the MST studies, fluorescently labelled TsaD = 10 nM, TsaB = 0.5 μM (10 × K_d) and TsaE = 20 μM (∼10 × K_d).

Figure 6.

Structural characteristics of the BK951–EcTsaBD complex. (A) Stereoview of the residual 2Fo-Fc electron density contoured at 1σ levels for the bound BK951 (green sticks) with metal ion (gray sphere) and surrounding side chains (red sticks). (B) Cartoon presentation of the TsaBD heterodimer bound to BK951; TsaB in blue, TsaD in red, BK951 in green sticks and Zinc as a grey sphere. (C) BK951 (green sticks) bound in the active site of TsaD, Zinc ion as grey sphere. H-bonds and ligand-metal interactions: black dotted lines. (D) LigPlot+ (69) representation of the inhibitor BK951 bound into the active site of EcTsaD (E) Superposition of ATPγS bound to StTsaD (orange sticks) onto BK951 bound to EcTsaD (green sticks). Only residues from EcTsaD are labelled.

Figure 7.

t⁶A activity of active site TsaD mutants using the full assay mixture (see Materials and Methods for details).

Figure 8.

Stability of TC-AMP bound to either EcTsaBD or EcTsaC. (A) Comparison of rate of decomposition of 10 μM TC-AMP at 37°C in 50 mM HEPES pH 7.5, 300 mM NaCl with 20 μM TsaBD (filled circles); 13 μM TsaC (filled diamonds); or in the absence of enzyme (filled triangles). (B) Concentration dependence of the rate of decomposition of a 1.3:1 molar ratio of TsaC:TC-AMP at varying concentrations under the same conditions as (A).

Figure 9.

Multiple sequence alignment of the TsaD/Kae1/OSGEP family members was realized using the blosum 45 with MAFFT (online version 7) (70). Structure-based alignment was performed using the ESPRIPT web-server (71). The SGG peptide conserved in all homologs is highlighted in pink.