Structural basis of the mercury(II)-mediated conformational switching of the dual-function transcriptional regulator MerR

Abstract

The mer operon confers bacterial resistance to inorganic mercury (Hg(2+)) and organomercurials by encoding proteins involved in sensing, transport and detoxification of these cytotoxic agents. Expression of the mer operon is under tight control by the dual-function transcriptional regulator MerR. The metal-free, apo MerR binds to the mer operator/promoter region as a repressor to block transcription initiation, but is converted into an activator upon Hg(2+)-binding. To understand how MerR interacts with Hg(2+) and how Hg(2+)-binding modulates MerR function, we report here the crystal structures of apo and Hg(2+)-bound MerR from Bacillus megaterium, corresponding respectively to the repressor and activator conformation of MerR. To our knowledge, the apo-MerR structure represents the first visualization of a MerR family member in its intact and inducer-free form. And the Hg(2+)-MerR structure offers the first view of a triligated Hg(2+)-thiolate center in a metalloprotein, confirming that MerR binds Hg(2+) via trigonal planar coordination geometry. Structural comparison revealed the conformational transition of MerR is coupled to the assembly/disassembly of a buried Hg(2+) binding site, thereby providing a structural basis for the Hg(2+)-mediated functional switching of MerR. The pronounced Hg(2+)-induced repositioning of the MerR DNA-binding domains suggests a plausible mechanism for the transcriptional regulation of the mer operon.

Figure 1.

Secondary and tertiary structures of apo and Hg²⁺-bound MerR. Ribbon representations of the MerR protomer in apo (A) and Hg²⁺-bound (B) states. For both structures, the DNA-binding domain, dimerization helix and metal-binding motif are in purple, orange and red, respectively. The H4–H5 loop is highlighted in blue. The apo and Hg²⁺-bound MerR structures were first aligned by superimposing on the backbone atoms of the dimerization helices (H5) before being displayed in separate panels. (C) Secondary structures of MerR in the apo and Hg²⁺-bound state. The Hg²⁺-ligating cysteine residues are marked cyan in the MerR sequence. The α-helices and β-strands suggested by the PredictProtein server (https://www.predictprotein.org/) are colored red and green in the sequence for comparison.

Figure 2.

Overall structures of apo and Hg²⁺-bound MerR homodimer. Ribbon representations of the MerR homodimer in apo (A) and Hg²⁺-bound (B) state. Two orthogonal views of each structure related by a 90° rotation about the coiled-coil dimerization region are provided. For both structures, the DNA-binding domain, dimerization helix and metal-binding motif of one subunit are in purple, orange and red, respectively, the second subunit is in gray. The H4–H5 loop is highlighted in blue. The two metal ions (Hg²⁺) in Hg²⁺-MerR are shown as yellow spheres. Labels belonging to the second protein subunit are flagged by a prime. The apo and Hg²⁺-bound MerR structures were first aligned by superimposing on the backbone atoms of the dimerization helices (H5 and H5’) before being displayed in separate panels.

Figure 3.

Detailed views of the Hg²⁺-induced movement of the metal-binding motif and assembly of the Hg²⁺ binding site. (A) Ribbon representation of apo-MerR homodimer (left) with an enlarged view (right) showing residues involved in the anchoring of the metal-binding motif as well as the spatial arrangement of the three Hg²⁺-ligating cysteine residues. (B) Ribbon representation of Hg²⁺-MerR homodimer (left) with an enlarged view (right) showing the coordination of Hg²⁺ (yellow sphere) by three cysteine residues and the involvement of H7 in Hg²⁺-binding. (C) Superimposition of apo-MerR (green) and Hg²⁺-MerR (cyan) shows that H7 and the three Hg²⁺-ligating cysteine residues undergo large repositioning upon Hg²⁺-binding. (D) The Hg²⁺-induced lengthening of H5 would result in steric clashes with the H5–H6 loop and H6 seen in apo-MerR structure (indicated by the orange arrowhead), causing conformational change in the metal-binding motif (highlighted by the red arrow). Labels belonging to the second protein subunit are flagged by a prime.

Figure 4.

The DNA-binding domains of MerR homodimer undergo Hg²⁺-induced repositioning. (A) Superimposition of apo-MerR (green) and Hg²⁺-MerR (cyan) shows the Hg²⁺-thiolate-tethered H7 in Hg²⁺-MerR would clash with H3 and H4 seen in apo-MerR (indicated by the orange arrowhead), which is expected to trigger the Hg²⁺-induced relocation of the DNA-binding domain. The DNA-binding domain is anchored by distinct set of stabilization interactions in apo-MerR (B) and Hg²⁺-MerR (C). Yellow sphere represents the bound Hg²⁺.

Figure 5.

MerR binds Hg²⁺ in a trigonal planar coordination geometry. (A) Structures and electron density map of the two crystallographically independent Hg²⁺ binding sites in the MerR homodimer; each Hg²⁺ (yellow sphere) is coordinated by three cysteine residues to form a trigonal-planar Hg²⁺-thiolate complex. The averaged Hg²⁺-S bond length (red dashed lines) is ∼2.4 Å. The 2mF_o-DF_c map (middle, purple meshes) and the anomalous difference maps (right, purple spheres) are contoured at 1.5 and 3/6 σ, respectively, above mean level. (B) The two Hg²⁺ binding sites are structurally similar and can be superimposed with an RMSD of 0.2 Å over all equivalent atom pairs. (C) Being surrounded by the pyrrolidine rings of Pro115 and Pro124 (indicated by the orange arrowheads), H3, H4, H7 and H5-H7 loop, the Hg²⁺-thiolate center is fully buried inside MerR and thus shielded from the solvent. Except for the N-terminus of H7, all other second shell Hg²⁺-contacting atoms are predominantly hydrophobic.

Figure 6.

Structural basis of the Hg²⁺-mediated transcriptional regulation of the mer operon by MerR. (A) A typical bacterial promoter with the −10 and −35 elements (colored in yellow) being spaced by 17±1 base pairs, which allows productive association with the σ subunit of the RNA polymerase holoenzyme. The σ2/σ3 (red) and σ4 (gray) domains of the σ subunit are modeled onto the −10 and −35 elements, respectively, based on the crystal structures of the binary complexes formed by σ subunit and DNA.