The structure and function of Mycobacterium tuberculosis MazF-mt6 toxin provide insights into conserved features of MazF endonucleases

Abstract

Toxin-antitoxin systems are ubiquitous in prokaryotic and archaeal genomes and regulate growth in response to stress. Escherichia coli contains at least 36 putative toxin-antitoxin gene pairs, and some pathogens such as Mycobacterium tuberculosis have over 90 toxin-antitoxin operons. E. coli MazF cleaves free mRNA after encountering stress, and nine M. tuberculosis MazF family members cleave mRNA, tRNA, or rRNA. Moreover, M. tuberculosis MazF-mt6 cleaves 23S rRNA Helix 70 to inhibit protein synthesis. The overall tertiary folds of these MazFs are predicted to be similar, and therefore, it is unclear how they recognize structurally distinct RNAs. Here we report the 2.7-Å X-ray crystal structure of MazF-mt6. MazF-mt6 adopts a PemK-like fold but lacks an elongated β1-β2 linker, a region that typically acts as a gate to direct RNA or antitoxin binding. In the absence of an elongated β1-β2 linker, MazF-mt6 is unable to transition between open and closed states, suggesting that the regulation of RNA or antitoxin selection may be distinct from other canonical MazFs. Additionally, a shortened β1-β2 linker allows for the formation of a deep, solvent-accessible, active-site pocket, which may allow recognition of specific, structured RNAs like Helix 70. Structure-based mutagenesis and bacterial growth assays demonstrate that MazF-mt6 residues Asp-10, Arg-13, and Thr-36 are critical for RNase activity and likely catalyze the proton-relay mechanism for RNA cleavage. These results provide further critical insights into how MazF secondary structural elements adapt to recognize diverse RNA substrates.

Keywords: X-ray crystallography; bacterial toxin; endoribonuclease; protein synthesis; ribosomal ribonucleic acid (rRNA) (ribosomal RNA); ribosome.

Figure 1.

Location of 23S rRNA Helix 70 in the context of the ribosome. A, overview of the 70S containing A-site, P-site, and E-site tRNAs (PDB code 4Y4R) (left panel). An expanded view of 23S rRNA Helix 69 (H69) and H70 and 16S rRNA helix 44 (h44) is shown in the right panel. H70 residues that MazF-mt6 targets are shown in red. B) H70 (nucleotide positions 1933–1967) indicating the location of the RT primer (blue), MazF-mt6 cleavage site (red arrowhead), and RT stop at nucleotide 1935. C, RT assays demonstrating MazF-mt6-induced 23S rRNA or H70 cleavage. The cleavage products were monitored by RT, and the reactions were run on an 8 m urea polyacrylamide sequencing gel. The MazF-mt6 full-length RT stop (FL), the major cleavage site (red arrowhead), and a minor cleavage site (black arrowhead) are indicated.

Figure 2.

Structure of M. tuberculosis MazF-mt6. A, the MazF-mt6 is a homodimer consisting of seven β-strands and four α-helices. B, A 90° rotated view to show the SH3-like antiparallel β-sheet and the proximity of the active-site residue location on the β1-β2 and β3-β4 linkers.

Figure 3.

MazF-mt6 residues important for activity. A, MazF-mt6 putative active site highlighting the residues that were targeted for single variant analysis. B and C, E. coli BW25113 growth monitored over 5 h postinduction of WT MazF-mt6 and active-site variants. Cells were grown in M9 medium supplemented with 0.2% glycerol and 0.2% casamino acids, and induction was initiated by the addition of 0.2% arabinose after cells reached an optical density of 0.2 at 600 nm. Error bars represent standard error of means from three independent experiments.

Figure 4.

MazF-mt6 residues Asp-10, Arg-13, and Thr-36 are critical for endonuclease activity. A, single-turnover experiments monitoring H70 cleavage by WT MazF-mt6 and MazF-mt6 variants. The products were analyzed on an 8 m urea polyacrylamide sequencing gel. The major cleavage product is denoted with a red arrowhead at U1940, and other minor cleavage products are indicated at positions 1941–1943. B, the product progression plot of WT MazF-mt6 and MazF-mt6 variants as monitored over a time course of 45 min. Error bars represent standard error of means from two independent experiments.

Figure 5.

Comparison of MazFs. A, comparison of electrostatic interactions between the EcMazF β1-β2 and β5-β6 linkers with an adjacent EcMazF dimer (PDB code 5CR2) (left) with the absence of electrostatic interactions between MazF-mt6 dimers (right). Residues responsible for stabilizing the β1-β2 linker in EcMazF are shown as sticks with dotted lines highlighting ionic bonds. The equivalent residues to EcMazF are shown for MazF-mt6, but there are no interactions between these residues. B, electrostatic surface potential of BsMazF with both the MazE C-terminal α-helix (magenta) and the mRNA (white) shown to emphasize regions where their binding overlaps (PDB code 4ME7) (left). The electrostatic surface potential of MazF-mt6 with both the putative antitoxin-binding path (dashed magenta α-helix) and the RNA-binding pocket (white circle) is shown. The putative antitoxin-binding path is negatively charged in contrast to BsMazF. C, homology model of MazE-mt6 C-terminal α-helix (blue) superimposed onto MazF-mt6. MazF-mt6 catalytic residue Arg-13 is predicted to be close to MazE-mt6 residues Asp-90, Asp-92, Asp-104, and Asp-106, which could potentially disorder the MazF-mt6 active site. The MazE-mt6 peptide sequence is shown below.

Figure 6.

Comparison of MazF nucleotide specificities. A, BsMazF active-site residues Gly-22, Arg-24, and Thr-48 interact with its mRNA substrate (PDB code 4MDX) (left panel). The BsMazF upstream and downstream RNA-binding regions with proposed key residues are indicated. B, the MazF-mt6 active-site residues Asp-10, Arg-13, and Thr-36 with a modeled RNA bound (outline). The equivalent MazF-mt6 upstream and downstream RNA-binding regions with potentially key residues are highlighted.

Figure 7.

Structural comparison of MazF-mt6 and CcdB toxin. A, E. coli CcdB toxin similarly forms a homodimer toxin (one monomer shown in green with the other shown in gray) with important secondary structural regions labeled (PDB code 1X75). B, two homodimers of MazF-mt6 are shown (one monomer shown in light gray with the other shown in dark gray) with the putative RNA-binding regions colored in red. C, a 90° rotated view of A of CcdB bound to GyrA (cyan) illustrating how CcdB α4 from both monomers engages its substrate, which is located on the opposite side of the RNA-binding domain of MazF-mt6. To bind GyrA, CcdB bends 12° (shown in dark gray). CcdB toxin in the absence of GyrA is shown for comparison (white).