Structural characterization of antibiotic self-immunity tRNA synthetase in plant tumour biocontrol agent

Abstract

Antibiotic-producing microbes evolved self-resistance mechanisms to avoid suicide. The biocontrol Agrobacterium radiobacter K84 secretes the Trojan Horse antibiotic agrocin 84 that is selectively transported into the plant pathogen A. tumefaciens and processed into the toxin TM84. We previously showed that TM84 employs a unique tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase (LeuRS), while the TM84-producer prevents self-poisoning by expressing a resistant LeuRS AgnB2. We now identify a mechanism by which the antibiotic-producing microbe resists its own toxin. Using a combination of structural, biochemical and biophysical approaches, we show that AgnB2 evolved structural changes so as to resist the antibiotic by eliminating the tRNA-dependence of TM84 binding. Mutagenesis of key resistance determinants results in mutants adopting an antibiotic-sensitive phenotype. This study illuminates the evolution of resistance in self-immunity genes and provides mechanistic insights into a fascinating tRNA-dependent antibiotic with applications for the development of anti-infectives and the prevention of biocontrol emasculation.

Figure 1. Biology of agrocin 84 produced by biocontrol A. radiobacter strain K84.

Pathogenic A. tumefaciens possesses a TiC58 plasmid that upon infection of the host plant makes the plant produce a carbon and phosphate source agrocinopine. The pathogen takes up agrocinopine via an agrocinopine permease encoded on pTiC58. This transporter also recognizes the uptake moiety on agrocin 84, an antibiotic produced by the plant tumour biocontrol, A. radiobacter strain K84. Upon entry of agrocin 84 into the pathogen, it is cleaved into the toxic moiety TM84 and the transport moiety. TM84 targets the leucyl tRNA synthetase, thereby inhibiting the aminoacylation of tRNA^Leu. This subsequently leads to the cessation of protein synthesis in the pathogen and leads to cell death. TM84 however has no effect on the aminoacylation reaction of a self-immunity LeuRS called AgnB2 that is encoded by the pAgK84 plasmid in A. radiobacter K84.

Figure 2. AgnB2 discriminates between a stable Leu-AMP analogue and TM84.

(a) Chemical structures of Leu-AMP and TM84 with differences highlighted in green. A non-hydrolysable adenylate analogue called Leu-AMS, which contains a N-sulfamoyl linkage in place of the phosphoanhydride of Leu-AMP, was used in biophysical and crystallography experiments. (b) Effect of TM84 and Leu-AMS on the aminoacylation reaction of wt AgnB2. Aminoacylation reactions were carried out using 2 nM wt AgnB2 only (●) and wt AgnB2 in the presence of 1 μM TM84 (■) and 20 nM Leu-AMS (▲) at 28 °C, pH 7.4 and initiated using 1 mM ATP. Error bars represent s.d. (n=3).

Figure 3. Structure of the AgnB2·tRNA^Leu·Leu-AMS complex.

(a) X-ray structure of wild-type AgnB2·tRNA^Leu·Leu-AMS complex with 3′-tRNA^Leu (blue) bound in the editing site (PDB Code—5AH5). The catalytic, editing, anticodon binding and C-terminal domains are displayed in yellow, cyan blue, red and gold, respectively. Leu-AMS bound to the synthetic active site is represented in a stick structure and the catalytic KQSKS and HIGH loops are highlighted in green. (b,c) Comparison of wt AgnB2·tRNA·Leu-AMS complex and LeuRS_Ec· tRNA^Leu·Leu-AMS complex binding site residues. The surface of the catalytic active site of the protein is depicted in yellow with Leu-AMS bound (stick structure).

Figure 4. Domain structure and primary sequence alignment of LeuRSs and AgnB2.

Domain structure of LeuRS and primary sequence alignment of the catalytic domain and LS domains of Thermus thermophilus LeuRS (LeuRS_Tt), LeuRS_Ec, LeuRS_At, plasmid encoded AgnB2 (from A. radiobacter strain K84) and LeuRS_Hp. The highly conserved regions are depicted in grey and the residues highlighted in bold (blue) represent the non-conserved residues in AgnB2. In addition, both AgnB2 and LeuRS_Hp lack the leucine-specific domain (pink) that is present in other LeuRSs.

Figure 5. Interactions of key residues in the catalytic active site of AgnB2.

Ribbon structure of the catalytic active site of (a) AgnB2·tRNA^Leu·Leu-AMS complex and (b) E. coli·tRNA^Leu·Leu-AMS complex. AgnB2 key residues interacting with Leu-AMS (shown as white sticks) or adjacent to the catalytic site are depicted as sticks with the following colour code: residues of the K⁵⁷²QSKS⁵⁷⁶ and of the H⁴⁷IGH⁵⁰ catalytic motifs, are coloured in green; core permissive residues T42 and A102 are shown in yellow; T581 which H-bonds to Q573 in pink; and D567 and R571 which forms the salt bridge mutated in this study and residue L577 are also in pink. E. coli LeuRS equivalent residues in the aminoacylation complex (PDB: 4AQ7) are shown in panel b. Key interactions in both the AgnB2 and the E. coli complexes are shown as red-dashed lines.

Figure 6. Comparison of antibiotic sensitivity of wt and mutant forms of LeuRS_At and AgnB2.

A. tumefaciens NTL4 T7pol-Gm-FRT (pYW15d-acs ΔaccR) strain transformed with pBBR1MCS-3 plasmids containing genes encoding wt or mutant LeuRS_At or AgnB2 constructs were tested for their sensitivity towards 50 μl of agrocin 84 (60 μM) placed in central well of a bioassay plate. For negative control, sterile ddH₂O was used instead of the antibiotic. (a) Bioassay results in graphical form indicating the sensitivity of wt LeuRS_At and LeuRS_At mutants; and (b) the resistance properties of wt AgnB2 and selected AgnB2 mutants; (c) Comparison of the TM84 inhibition of the aminoacylation reactions of wt AgnB2 (●), AgnB2 Triple Q413R mutant (■) and AgnB2 Triple LS domain mutant (▲). Dose–response curves and the resulting IC₅₀ values were obtained at saturating leucine and (0.675 mM) ATP concentrations (see Methods). Error bars represent s.d. from three independent experiments.

Figure 7. The accumulative mutation of tRNA interacting elements with core Triple mutations in AgnB2 increases the enzyme's affinity to TM84.

ITC results depicted in graphical form compare the TM84-binding affinities of wt AgnB2 and Triple AgnB2 mutant±Q413R or LS domain to (a) TM84 alone; or (b) TM84 in the presence of tRNA^Leu. Error bars represent standard deviation from three independent experiments.

Figure 8. Model illustrating determinants of TM84 sensitivity and resistance in LeuRS_At and AgnB2.

The ternary complex of LeuRS_At forms a tight binding complex with the CCA 3′-end of the tRNA and TM84. Upon mutation of three critical residues, P45, M633 and N105, a TM84 resistant mutant (LeuRS_At Triad) is obtained. AgnB2 on the other hand forms a weak ternary complex with tRNA and TM84, thereby explaining its high level of resistance towards TM84. Mutagenesis of the corresponding residues in AgnB2 with the residues in LeuRS_At results in the AgnB2 Triple mutant that has reduced levels of AgnB2 resistance. Upon introduction of Q413R mutation or addition of a LS-domain to the AgnB2 triple mutant, increased sensitivity towards TM84 is achieved. Thus these residues along with the LS domain may be the key determinants of sensitivity or resistance in LeuRS_At and AgnB2.