Structure of the periplasmic copper-binding protein CueP fromSalmonella entericaserovar Typhimurium

Abstract

CueP was initially identified as a copper-resistance gene in Salmonella enterica serovar Typhimurium, which has evolved to survive in the phagosomes of macrophages. Recently, CueP was determined to be a periplasmic copper-binding protein and has been implicated in the transfer of copper ions to SodCII in the periplasm. In this study, the crystal structure of CueP has been determined, revealing a V-shaped dimeric structure. The conserved cysteine and histidine residues are clustered on the surface of one side of the C-terminal domain, suggesting that this cysteine- and histidine-rich region is related to the function of CueP. LC-MS/MS analysis established the presence of a disulfide bond between Cys96 and Cys176 under aerobic conditions. Subsequent biophysical analyses showed that the CueP protein binds copper and zinc, and the mutation of Cys104 to serine (C104S) dramatically reduced the binding affinity for copper and zinc, suggesting that the cysteine- and histidine-rich cluster is responsible for copper binding. This study provides a structural basis for the participation of CueP in the resistance of the intracellular pathogen Salmonella to copper.

Compartment and signal-specific codependence in the transcriptional control of Salmonella periplasmic copper homeostasis

“…S5), reflecting a conserved selective pressure to maintain the coordinated expression control of these xenolog proteins during evolution. CueP is a copper-binding protein (14,16) required for resistance to the metal ion (8). Its presence alleviates copper stress (25) and reduces the cellular content of the ion (15).…”

“…As a Cu-binding protein (14,16), the unregulated expression of CueP would restrict the availability of the metal ion for other factors. Nevertheless, attempts to determine differences in the copper content of periplasmic cuproproteins like SodCII at each growth time point were unsuccessful, likely owing to a lack of sensitivity.…”

“…Different lines of evidence suggest that the CueR-induced periplasmic protein (CueP), product of the cueP gene, plays a major role in maintaining copper homeostasis in the Salmonella envelope. It participates in copper resistance, particularly in anaerobiosis (8,(14)(15)(16), and cueP is usually present in species lacking the cus locus (8).…”

Bacterial Copper Resistance and Virulence

“…Recently, CueP from Salmonella enterica serovar Typhimurium was shown to be required for the metallation of SodCII [35]. The crystal structure of CueP has been determined revealing a V-shaped dimeric structure [36] with no homology to other known Cu chaperones.…”

Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA

“…For identification and quantification of peptides modified by 2-NBA methyl ester or iodoacetamide, monomer bands of wild-type NbaA and the Tyr193Phe mutant were isolated from nondenaturing SDS-PAGE gels digested with both trypsin and chymotrypsin in order to improve the elution and detection rate of the modified peptide(s) from the nLC-tandem mass spectrometry, as described previously (26). All samples were prepared under N 2 gas and stored in dark vials at 4°C to avoid artifacts that arise from cysteine oxidation.…”