Selective cadmium regulation mediated by a cooperative binding mechanism in CadR.

Detoxification of the highly toxic cadmium element is essential for the survival of living organisms. Pseudomonas putida CadR, a MerR family transcriptional regulator, has been reported to exhibit an ultraspecific response to the cadmium ion. Our crystallographic and spectroscopic studies reveal that the extra cadmium selectivity of CadR is mediated by the unexpected cooperation of thiolate-rich site I and histidine-rich site II. Cadmium binding in site I mediates the reorientation of protein domains and facilitates the assembly of site II. Subsequently, site II bridge-links 2 DNA binding domains through ligands His140/His145 in the C-terminal histidine-rich tail. With dynamic transit between 2 conformational states, this bridge could stabilize the regulator into an optimal conformation that is critical for enhancing the transcriptional activity of the cadmium detoxification system. Our results provide dynamic insight into how nature utilizes the unique cooperative binding mechanism in multisite proteins to recognize cadmium ions specifically.

A simplified protein purification method through nickel cleavage of the recombinant protein from the Escherichia coli cell surface.

To simplify the protein purification process, we developed a novel one-step purification method in which the recombinant protein can be cleaved directly from the Escherichia coli cell surface. This method involves fusion of the target protein to the C-terminus of a LOS tag comprising a surface anchor protein (Lpp-OmpA) and a sequence-specific nickel-assisted cleavage (SNAC)-tag. The LOS tag facilitates the anchoring of the target protein to the outer membrane of E. coli cells and its separation from the cell membrane through Ni2+ cleavage. Intact, biologically active protein with a purity of 95% and a yield of approximately 100 mg per liter of culture can be readily obtained through Ni2+ cleavage in resuspension solution followed by centrifugation. In this study, a practical and promising protein purification method has been established with minimal labor and cost, as no cell disruption and chromatographic separation are required downstream.

Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/ß-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved ß-sheets pocket. The dimerization of the N-terminal H1 and H1' α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents.

Structural Basis for the Selective Pb(II) Recognition of Metalloregulatory Protein PbrR691.

The transcription regulator PbrR691, one of the MerR family proteins, shows extremely high sensitivity and selectivity toward Pb(II) in Ralstonia metallidurans CH34. Here, we present the crystal structure of PbrR691 in complex with Pb(II) at 2.0 Å resolution. The Pb(II) coordinates with three conserved cysteines and adopts a unique trigonal-pyramidal (hemidirected) geometry. To our knowledge, the PbrR691-Pb(II) structure provides the first three-dimensional visualization of a functional hemidirected lead(II) thiolate coordinate geometry in a protein.

Directed evolution of the genetically encoded zinc(II) FRET sensor ZapCY1.

Zinc(II) ions (Zn2+) play an essential role in living systems, with their delicate concentration balance differing among the various intracellular organelles. The spatiotemporal distribution and homeostasis of Zn2+ can be monitored through photoluminescence imaging using zinc sensors. Among such biosensors, genetically encoded fluorescent sensor proteins are attractive tools owing to their subcellular localization advantage and high biocompatibility. However, the limited fluorescent properties of these proteins, such as their insufficient quantum yield and dynamic range, restrict their practical use. In this study, we developed an expression-screening-directed evolution system and used it to improve ZapCY1, a genetically encoded fluorescence resonance energy transfer (FRET) sensor. After four rounds of directed evolution, the FRET dynamic range of the modified sensor (designated ZapTV-EH) was increased by 1.5-1.7-fold. With its enhanced signal-to-noise ratio and ability to detect a wide Zn2+ concentration range, ZapTV-EH proves to be a better visualization tool for monitoring Zn2+ at the subcellular level. Combined with the simplified subcloning and expression steps and sufficient mutant libraries, this directed evolution system may provide a more simple and efficient way to develop and optimize genetically encoded FRET sensors through high-throughput screening.

Temperature-Driven Metalloprotein-Based Hybrid Hydrogels for Selective and Reversible Removal of Cadmium(II) from Water.

To develop biomaterials that easily and reversibly remove trace amounts of metal ions, we synthesized PNIPAM-co-CadRP, a thermally sensitive hybrid hydrogel by immobilizing a reconstituted cadmium binding peptide (CadRP) derived from the metalloregulatory protein CadR in a poly(N-isopropylacrylamide) (PNIPAM) gel network. The hybrid hydrogel retains the properties of the immobilized peptide and highly sensitively and selectively binds Cd(II) ions. The thermally sensitive properties of the hybrid hydrogel, which swells at low temperatures (<34 °C) and shrinks at high temperatures, provides a driving force sufficient to alternate the conformation of the immobilized CadRP such that the peptide captures and releases metal ions at high and low temperatures, respectively. Using this novel hybrid gel, we captured nanomolar Cd(II) from samples of environmental water in a highly efficient manner, leading to a practical and repeatedly reusable material to remediate our environment.

Structural Analysis of the Hg(II)-Regulatory Protein Tn501 MerR from Pseudomonas aeruginosa.

The metalloprotein MerR is a mercury(II)-dependent transcriptional repressor-activator that responds to mercury(II) with extraordinary sensitivity and selectivity. It's widely distributed in both Gram-negative and Gram-positive bacteria but with barely detectable sequence identities between the two sources. To provide structural basis for the considerable biochemical and biophysical experiments previously performed on Tn501 and Tn21 MerR from Gram-negative bacteria, we analyzed the crystal structure of mercury(II)-bound Tn501 MerR. The structure in the metal-binding domain provides Tn501 MerR with a high affinity for mercury(II) and the ability to distinguish mercury(II) from other metals with its unique planar trigonal coordination geometry, which is adopted by both Gram-negative and Gram-positive bacteria. The mercury(II) coordination state in the C-terminal metal-binding domain is transmitted through the allosteric network across the dimer interface to the N-terminal DNA-binding domain. Together with the previous mutagenesis analyses, the present data indicate that the residues in the allosteric pathway have a central role in maintaining the functions of Tn501 MerR. In addition, the complex structure exhibits significant differences in tertiary and quaternary structural arrangements compared to those of Bacillus MerR from Gram-positive bacteria, which probably enable them to function with specific promoter DNA with different spacers between -35 and -10 elements.