Fig 1.
Schematic diagram of the CzcR-CzcS TCS in P. aeruginosa, overall structure of CzcS-Zn, and superimpositions of CzcS SD with other PDC members.
(a) A schematic diagram of the CzcR-CzcS TCS in P. aeruginosa. The CzcR-CzcS TCS is involved in regulating Zn(II) resistance, antibiotic resistance, quorum sensing, and virulence factors. The periplasmic sensor domain of the HK CzcS is indicated with the blue box. (b) Overall structure of CzcS-Zn. The CzcS SD are shown as cartoon diagrams with labelled secondary structures. The coordinated Zn(II) is depicted as sphere in red. (c) Superimpositions of the CzcS SD with other PDC members in tertiary structure. All structures are shown as cartoon representations with the CzcS SD colored in yellow in all panels. The PhoQ SD (PDB code: 3BQ8), CitA SD (PDB code: 2J80), and DcuS SD (PDB code: 3BY8) are colored in magenta, green, and cyan, respectively. The citrate and malate ligands, which are bound to the central β-sheets, are shown as spherical representations in CitA and DcuS, respectively. The pairwise structure comparisons are performed by the PDBefold [17].
Table 1.
Data collection and refinement statistics for the structure of CzcS-Zn.
Fig 2.
The Zn(II) binding site of CzcS SD.
(a) The coordination environment of the functionally relevant Zn(II) ion. The Zn(II) ion is shown as sphere in red, and the coordinated residues are shown as sticks in atomic color (C, yellow; N, blue; O, red). The 2Fo-Fc omit map is contoured at the 1σ level and is colored in gray. (b) The detailed interactions between the Zn(II) and the coordinated residues. The Zn(II) binds to CzcS in the tetrahedral coordination geometry. The Zn(II) ion is shown as red sphere. The residues in the first coordination sphere are depicted as stick representations with Cα atoms in yellow. The second shell hydrogen-bond interactions are indicated by the magenta-dotted line. (c) Metal and antibiotic tolerance plate assay. Wild type P. aeruginosa and its derivative strains are examined on LB plates that contain Zn(II) and MEPM antibiotic as follows: wild type P. aeruginosa with empty pAK1900 plasmid as the control (WT PAO1 pAK1900), czcS-deficient P. aeruginosa with the empty pAK1900 (PAO1△czcS pAK1900), czcS-deficient P. aeruginosa with wild type czcS encoded on pAK1900 (PAO1△czcS pCSAK), czcS-deficient P. aeruginosa complemented with the czcS mutants in pAK1900 (PAO1△czcS pCSAK H55A, PAO1△czcS pCSAK H55C, PAO1△czcS pCSAK D60A, PAO1△czcS pCSAK D60C, and PAO1△czcS pCSAK H55R).
Fig 3.
The identification of Co(II)-responsive mutant.
Wild type P. aeruginosa and its derivative strains are examined on LB plates that contain Co(II) and MEPM antibiotic as follows: wild type P. aeruginosa with empty pAK1900 plasmid as the control (WT PAO1 pAK1900), czcS-deficient P. aeruginosa with the empty pAK1900 (PAO1△czcS pAK1900), czcS-deficient P. aeruginosa with wild type czcS encoded on pAK1900 (PAO1△czcS pCSAK), czcS-deficient P. aeruginosa complemented with the czcS mutants in pAK1900 (PAO1△czcS pCSAK D60C).
Fig 4.
Cysteine substitution of residues in the linker region.
(a) The schematic diagram of the linker region in the HK CzcS. The linker region, which connects the H1 and H1’ α-helices of the sensor domain to the transmembrane helices, is indicated by dashed lines. The amino acid sequence in the linker region is “Arg-Glu-Leu-Glu”. (b) Metal and antibiotic tolerance plate assay. Wild type P. aeruginosa and its derivative strains are examined on LB plates that contain Zn(II) and MEPM antibiotic as follows: wild type P. aeruginosa with the empty pAK1900 plasmid as the control (WT PAO1 PAK1900), czcS-deficient P. aeruginosa with empty pAK1900 (PAO1△czcS pAK1900), czcS-deficient P. aeruginosa with wild type czcS encoded on pAK1900 (PAO1△czcS pCSAK), czcS deficient P. aeruginosa complemented with czcS mutants in pAK1900 (PAO1△czcS pCSAK H55A, and PAO1△czcS pCSAK L38C H55A).
Fig 5.
Proline substitution of residues along the H1 and H1’ α-helices.
(a) The schematic diagram showing the locations of the residues involved in the proline substitutional experiments. The positions of Gln52, Leu50, Leu48, Asn45, Arg43, and Arg41 on the H1 and H1’ α-helices are labelled with circles in different colors. (b) Metal and antibiotic tolerance plate assay of single proline substitutions. Wild type P. aeruginosa and its derivative strains are examined on the LB plates that contain Zn(II) and MEPM antibiotic as follows: wild type P. aeruginosa with the empty pAK1900 plasmid as the control (WT PAO1 pAK1900), czcS deficient P. aeruginosa with empty pAK1900 (PAO1△czcS pAK1900), czcS-deficient P. aeruginosa with wild type czcS encoded on pAK1900 (PAO1△czcS pCSAK), czcS-deficient P. aeruginosa complemented with czcS mutants in pAK1900 (PAO1△czcS pCSAK Q52P, PAO1△czcS pCSAK L50P, PAO1△czcS pCSAK L48P, PAO1△czcS pCSAK N45P, PAO1△czcS pCSAK R43P, and PAO1△czcS pCSAK R41P).
Fig 6.
Proposed molecular mechanism of Zn(II) signal transduction in HK CzcS.
The two monomers in the CzcS functional dimer are shown in green and in cyan. When the Zn(II) binds to CzcS, the sensor domain will turn from monomer to dimer with the Zn(II) binding at the symmetrical N-terminal α-helices. The dimerization of sensor domain will drive the interactional rearrangement within the dimeric four-helical bundles in the transmembrane domain [47], and autophosphorylation is activated at the conserved histidine residues in the cytoplasmic kinase domain [48–51].