Interaction of Mycobacterium tuberculosis elongation factor Tu with GTP is regulated by phosphorylation

Abstract

During protein synthesis, translation elongation factor Tu (Ef-Tu) is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. The activity of Ef-Tu is dependent on its interaction with GTP. Posttranslational modifications, such as phosphorylation, are known to regulate the activity of Ef-Tu in several prokaryotes. Although a study of the Mycobacterium tuberculosis phosphoproteome showed Ef-Tu to be phosphorylated, the role of phosphorylation in the regulation of Ef-Tu has not been studied. In this report, we show that phosphorylation of M. tuberculosis Ef-Tu (MtbEf-Tu) by PknB reduced its interaction with GTP, suggesting a concomitant reduction in the level of protein synthesis. Overexpression of PknB in Mycobacterium smegmatis indeed reduced the level of protein synthesis. MtbEf-Tu was found to be phosphorylated by PknB on multiple sites, including Thr118, which is required for optimal activity of the protein. We found that kirromycin, an Ef-Tu-specific antibiotic, had a significant effect on the nucleotide binding of unphosphorylated MtbEf-Tu but not on the phosphorylated protein. Our results show that the modulation of the MtbEf-Tu-GTP interaction by phosphorylation can have an impact on cellular protein synthesis and growth. These results also suggest that phosphorylation can change the sensitivity of the protein to the specific inhibitors. Thus, the efficacy of an inhibitor can also depend on the posttranslational modification(s) of the target and should be considered during the development of the molecule.

Fig. 1.

Role of PknB in the regulation of protein synthesis and the phosphorylation of MtbEf-Tu in M. smegmatis. (A) Graphical representation of [³⁵S]Met incorporation into proteins of M. smegmatis cells overexpressing PknB or PknB^K40M. The PknB-overexpressing strain showed ∼25% less incorporation of [³⁵S]Met than the PknB^K40M-overexpressing strain when equal concentrations of the lysates were taken. The experiment was repeated four times; error bars represent standard deviations of four individual readings. (B) His₁₂-MtbEf-Tu was purified from M. smegmatis and was phosphoenriched. The enriched protein was assessed for phosphorylation by immunoblotting with anti-pThr antibodies. Lane 1, phosphoenriched sample; lane 2, PknB_c (positive control); lane 3, GST (negative control). (C) The phosphoenriched sample of MtbEf-Tu purified from M. smegmatis was also probed with anti-MtbEf-Tu antibodies. Lane 1, MtbEf-Tu^phos (positive control); lane 2, GST (negative control); lane 3, phosphoenriched sample. (D) Autoradiogram showing the in vitro phosphorylation of MtbEf-Tu (5 μg) by PknB_c (0.5 μg) in kinase buffer containing 2 μCi [γ-³²P]ATP. Samples were separated by 12% SDS-PAGE and were analyzed with a phosphorimager.

Fig. 2.

Phosphorylation of MtbEf-Tu and identification of phosphorylation sites. (A) MtbEf-Tu was coexpressed with either PknB or PknB^K40M in E. coli, and the phosphorylation status of purified MtbEf-Tu was analyzed by Western blotting using anti-pThr antibodies. (Top) Ponceau-stained nitrocellulose membrane; (bottom) immunoblot. Lane 1, PstP_c (negative control); lane 2, PknB_c (positive control); lane 3, MtbEf-Tu coexpressed with PknB^K40M (MtbEf-Tu^unphos)); lane 4, MtbEf-Tu coexpressed with PknB (MtbEf-Tu^phos); lane 6, molecular size markers. (B) Phosphorylated species in MtbEf-Tu^unphos (left) and MtbEf-Tu^phos (right) were separated by bidirectional PAGE, and the proteins were transferred to a nitrocellulose membrane and were blotted with anti-pThr (α-pThr) and anti-MtbEf-Tu antibodies. Additional spots were probed for MtbEf-Tu^phos, while no such spots were observed with MtbEf-Tu^unphos. (C) Phosphoamino acid analysis of MtbEf-Tu phosphorylated by PknB_c was performed by 2D-TLE and autoradiography. Mainly a Thr residue(s) was observed to be phosphorylated; no spot corresponding to pSer or pTyr was observed. (D) Diagrammatic representation of residues of MtbEf-Tu^phos phosphorylated by PknB during coexpression in E. coli. A probable domain-wise distribution of phosphorylation sites is shown.

Fig. 3.

Interaction of MtbEf-Tu with BODIPY FL-GTP. (A) The interactions of MtbEf-Tu^phos and MtbEf-Tu^unphos (2 μM each) with the fluorophore BODIPY FL-GTP (10 nM) were studied by recording the emission spectrum from 490 nm to 600 nm after excitation at 488 nm. A buffer containing BODIPY FL-GTP served as a control. (B) Spectrum recorded from 490 to 600 nm by adding increasing concentrations of MtbEf-Tu^unphos (1 to 10 μM) to a buffer containing BODIPY FL-GTP (10 nM). (C) Saturation curve for the MtbEf-Tu^unphos–BODIPY FL-GTP interaction. (D) Spectrum recorded from 490 to 600 nm by adding increasing concentrations of MtbEf-Tu^phos (1 to 10 μM) to a buffer containing BODIPY FL-GTP (10 nM). (E) Saturation curve for the MtbEf-Tu^phos–BODIPY FL-GTP interaction.

Fig. 4.

Phosphorylation and interaction of MtbEf-Tu^T118A with BODIPY FL-GTP. (A) Bar graph showing partial loss of the phosphorylation signal on the MtbEf-Tu^T118A mutant compared to the native protein when phosphorylated by PknB_c. (B and C) Interaction of increasing concentrations of MtbEf-Tu (B) or MtbEf-Tu^T118A (C) (1 μM to 4 μM) with a constant concentration of BODIPY FL-GTP (200 nM).

Fig. 5.

Dissociation of BODIPY FL-GDP from MtbEf-Tu and effect of Ef-Ts. Spontaneous dissociation of BODIPY FL-GDP from MtbEf-Tu (1 μM) was measured by titrating the MtbEf-Tu–BODIPY FL-GDP complex with an excess of unlabeled GDP (500 nM) at 25°C. BODIPY FL-GDP was excited at 488 nm, and emission was measured at 512 nm. (A) The dissociation of BODIPY FL-GDP from MtbEf-Tu^phos (curve 2), MtbEf-Tu^unphos (curve 3), and buffer F, used as a control (curve 1), was measured up to 800 s. (B) The dissociation of BODIPY FL-GDP from MtbEf-Tu^phos (curve 2) and MtbEf-Tu^unphos (curve 3) was measured in the presence of Ef-Ts (200 nM).

Fig. 6.

Effect of kirromycin on the interaction of MtbEf-Tu with GTP. The interactions of MtbEf-Tu^unphos (1 μM) (A) and MtbEf-Tu^phos (1 μM) (B) with BODIPY FL-GTP (10 nM) were studied in the presence of kirromycin (10 μM) by recording the emission spectrum from 500 to 600 nm.