Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

Abstract

Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78-Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

Keywords: crystal structure; persistence; ribonuclease; stress response; structure-function; substrate specificity; toxin-antitoxin.

FIGURE 1.

Structure of wild type EcMazF and EcMazF^E24A. A, amino acid sequence of EcMazF with the different secondary structure elements identified. Red arrows correspond to β-strands, and yellow rectangles correspond to α-helices. B, stereo view of the EcMazF dimer (PDB entry 5CR2) with each secondary structure element as well as the N and C termini labeled in one monomer. C, Cα trace of the EcMazF dimer of the d(AUACAUA) complex (PDB entry 5CR2) with one monomer in cyan and the other in green in two perpendicular orientations. Superimposed are all crystallographically independent monomers of the wild type EcMazF structures (in black) and of the EcMazF^E24A structures (in red). One EcMazF^E24A monomer of the complex with EcMazE(68–82) is shown in yellow. The position of loops β1-β2 and β4-β5 are indicated.

FIGURE 2.

Substrate binding mode. A, schematic representation of the EcMazF dimer (chains A and B in PDB entry 5CR2) with the bound oligonucleotides shown as yellow sticks. The three loops and the short helix α1 that form the binding site are highlighted in green (loop β1-β2, Ile-13–Ala-31), pink (α-helix, Phe-37–Gly-44), cyan (loop β3-β4, Thr-52–Phe-60), and orange (loop β4-β5, Leu-64–Gly-71). B, equivalent schematic representation of the BsMazF substrate complex (PDB entry 4MDX). In this structure, only one of the two substrate-binding sites is occupied. C, electron density for the substrate-mimicking DNA d(AUACAUA) observed bound to chain A of EcMazF. Only dU² up to dU⁶ are visible. D, surface representation of the substrate-binding site of EcMazF colored as in A. The exposed surfaces of Leu-64, Gln-67, Val-72, Leu-74, Asp-76, and Gln-77, which form the “bottom” of the upstream subsite and downstream-binding groove and do not contact the substrate directly, are colored red. The substrate-mimicking DNA 5′-AUACAUA-3′ is represented as sticks. Upstream and downstream binding sites are indicated. E, equivalent representation of 5′-UUdUACAUAA-3′ bound to BsMazF. Here, additional specificity-determining contacts are made between the oligonucleotide and the upstream and downstream binding sites, leading to a better complementarity of the surfaces of both macromolecules. F, schematic representation of nucleotide-specific recognition of d(AUACAUA) substrate by EcMazF. The substrate-mimicking DNA 5′-AUACAUA-3′ is represented as sticks. Black lines indicate hydrogen bonds, and red lines indicate hydrophobic contacts. Asterisks designate interactions with main chain atoms of the given EcMazF amino acid. No electron density is seen for dA¹ and dA⁷, and hence no interactions with the protein can be deduced. The extended conformation shown for the oligonucleotide does not represent the conformation of the molecule in the crystal, but is intended as a schematic. G, equivalent representation for the 5′-dUACAU-3′ moiety in the BsMazF complex (PDB entry 4MDX).

FIGURE 3.

Subsite details. A, details of the interactions in the upstream U binding cleft. Residues of EcMazF interacting with the base are shown in stick representation and colored as in Fig. 1A. The uridine base is surrounded by the aliphatic/aromatic parts of the side chains of Trp-14, Arg-29, Pro-30, Thr-52, Thr-53, and Arg-69. The specificity-determining hydrogen bond from the main chain NH of Arg-69 to O4 of the uridine base is shown as a gray dotted line. Amino acid side chains that are part of the bottom of the binding site but do not touch the substrate are colored red. The equivalent residues of BsMazF are superimposed as black sticks and labeled in black. B, details of the interactions in the downstream ACA-binding groove. Residues of EcMazF interacting with d(A³C⁴A⁵U⁶) are shown in stick representation and colored as in Fig. 1A. Corresponding secondary structure elements are shown as a schematic. Hydrogen bonds are represented as dotted lines. Amino acid side chains that are part of the bottom of the binding groove but do not touch the substrate are colored red.

FIGURE 4.

Possible catalytic mechanism for MazF. A, interactions with the catalytic residues. Stick representation of the d(U²A³) dinucleotide as found in the EcMazF complex is shown but with the O2′ of U² added to its most likely position. The likely hydrogen bonding network with this minimal substrate unit is shown as dashed black lines. B, proposed catalytic mechanism. Arg-29 acts as simultaneous general acid and general base by relaying a proton from O2′ of U2 to O5′ of A3 via a Grotthuss-like mechanism. Thr-52 likely contributes to catalysis by stabilizing the buildup of negative charge on the phosphate in the bipyramidal transition state.

FIGURE 5.

Inhibition of EcMazF by EcMazE(68–82). A, hydrogen bonding around Glu-24. In the EcMazF-d(AUACAUA) complex, the side chain of Glu-24 does not interact with the ligand. Rather, this side chain is buried, and its negative charge is neutralized by salt bridges with the side chains of Arg-86 and Lys-79. The EcMazF is represented as a schematic with one subunit colored green and the other cyan. B, electron density for the EcMazE(68–82) peptide. The different amino acid residues are labeled. C, superposition of the EcMazF dimers as seen in the d(AUACAUA) complex (green and cyan as in A) and the EcMazE(68–82) complex (yellow). The EcMazE(68–82) is shown in red. Relevant amino acid side chains are shown as sticks and are labeled. Glu-80 of EcMazE(68–82) in the peptide complex takes over the role of Glu-24 of EcMazF in the d(AUACAUA) complex.

FIGURE 6.

Isothermal titration calorimetry of EcMazE fragments binding to EcMazF. A, titration of EcMazE(50–82) into EcMazF^E24A. B, titration of EcMazE(54–77) into EcMazF^E24A. C, titration of EcMazE(68–82) into EcMazF^E24A. D, titration of EcMazE(50–82) into wild type EcMazF. E, titration of EcMazE(54–77) into wild type EcMazF. All the experiments were done at 305 K in 50 mm phosphate buffer, pH 7.0, 150 mm NaCl, 1 mm EDTA. Each time, the raw ITC data are shown in the upper panel, and the corresponding binding isotherm obtained from integrating the area under the peaks is shown in the lower panel.

FIGURE 7.

Parallels between EcMazF and CcdB. A, surface of the EcMazF dimer in the complex with EcMazE(68–82). The two EcMazF monomers are shown in different shades of blue. The EcMazE(68–82) peptide is shown in a yellow schematic representation, with the side chains of Trp-73 and Trp-82 highlighted. B, stereo view of the hydrophobic pocket accommodating EcMazE Trp-73 and Trp-82. The side chains of Val-15, Arg-29, Ala-31, Cys-48, Pro-50, Gln-77, Lys-79, and Ile-81 of EcMazF (chain A of PDB entry 5CQX) are shown in stick representation colored by atom type. The local backbone conformation is shown in schematic representation. The side chain of Trp-73 of EcMazE(68–82), which docks into this pocket, is shown in green. The side chain of EcMazE(68–82) Trp-82 (resulting from a superposition of chain B on chain A of the EcMazE(68–82) complex) is shown in cyan. EcMazF residue Phe-17 (superimposed from the d(AUACAUA) complex) is shown in yellow. C, surface of the F-plasmid CcdB dimer in its CcdA-bound conformation (PDB entry 3HPW). The two CcdB monomers are shown in different shades of blue and are aligned with the EcMazF dimer in A. A yellow schematic is shown for the CcdA peptide Phe-65–Trp-72, and the side chains of Phe-65–Trp-72 are highlighted. D, stereo representation of the superposition of the Cα traces of the EcMazF dimer in its EcMazE(68–82)-bound conformation (green and cyan) and its d(AUACAUA)-bound conformation (yellow). The bound d(AUACAUA) is shown in gray to identify the substrate binding region. Only one monomer was used to calculate the superposition. A clear relative rigid body displacement of both monomers relative to each other is seen, which moves helix α1 relative to the other three loops that together form the substrate-binding site.